Actrii proteins and use in treating post-capillary pulmonary hypertension

ABSTRACT

In some aspects, the disclosure relates to compositions and methods comprising ActRII polypeptides to treat, prevent, or reduce the progression rate and/or severity of post-capillary pulmonary hypertension (PcPH), particularly treating, preventing or reducing the progression rate and/or severity of one or more PcPH-associated complications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority from U.S. ProvisionalApplication No. 63/016,942, filed on Apr. 28, 2020 and from U.S.Provisional Application No. 63/159,253, filed on Mar. 10, 2021. Theforegoing applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Pulmonary hypertension (PH) is a disease characterized by high bloodpressure in lung vasculature, including pulmonary arteries, pulmonaryveins, and pulmonary capillaries. In general, PH is defined as a meanpulmonary arterial pressure (mPAP)≥20 mm Hg at rest or ≥30 mm Hg withexercise [Hill et al., Respiratory Care 54(7):958-68 (2009)]. One of themain PH symptoms is difficulty in breathing or shortness of breath, andother symptoms include fatigue, dizziness, fainting, peripheral edema(swelling in foot, legs or ankles), bluish lips and skin, chest pain,angina pectoris, light-headedness during exercise, non-productive cough,racing pulse and palpitations. PH can be a severe disease causing heartfailure, which is one of the most common causes of death in people whohave pulmonary hypertension. Postoperative pulmonary hypertension maycomplicate many types of surgeries or procedures, and present achallenge associated with a high mortality.

PH may be grouped based on different manifestations of the diseasesharing similarities in pathophysiologic mechanisms, clinicalpresentation, and therapeutic approaches [Simonneau et al., JACC 54(1):S44-54 (2009)]. Clinical classification of PH was first proposed in1973, and a recent updated clinical classification was endorsed by theWorld Health Organization (WHO) in 2018. According to the updated PHclinical classification, there are five main groups of PH: pulmonaryarterial hypertension (PAH), characterized by a pulmonary arterial wedgepressure (PAWP)≤15 mm Hg; PH due to left heart disease (also known aspulmonary venous hypertension or congestive heart failure),characterized by a PAWP>15 mm Hg; PH due to lung diseases and/orhypoxia; PH due to pulmonary artery obstructions; and PH with unclearand/or multifactorial mechanisms [Simonneau (2019) Eur Respir J:53:1801913]. PH due to left heart disease is further classified into PHdue to heart failure with preserved left ventricular ejection fraction;PH due to heart failure with reduced left ventricular ejection fraction;valvular heart disease; and congenital/acquired cardiovascularconditions leading to post-capillary PH [Simonneau (2019) Eur Respir J:53:1801913]. Diagnosis of various types of PH typically requires aseries of tests.

In general, PH treatment depends on the cause or classification of PH.Where PH is caused by a known medicine or medical condition, it is knownas a secondary PH, and its treatment is usually directed at theunderlying disease. Treatment of Group 2 pulmonary hypertension (e.g.,venous hypertension) generally involves optimizing left ventricularfunction by administering diuretics, beta blockers, angiotensinreceptor-neprilysin inhibitors (ARNI), and ACE inhibitors, cardiacresynchronization therapy, or repairing or replacing a mitral valve oraortic valve.

There is a high, unmet need for effective therapies for treatingpulmonary hypertension. Accordingly, it is an object of the presentdisclosure to provide methods for treating, preventing, or reducing theprogression rate and/or severity of PH, particularly treating,preventing or reducing the progression rate and/or severity of one ormore PH-associated complications.

SUMMARY OF THE INVENTION

In some embodiments, the disclosure provides for a method of treatingpost-capillary pulmonary hypertension (PcPH), comprising administeringto a patient in need thereof an effective amount of a polypeptidecomprising an amino acid sequence that is at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to an amino acid sequence that begins at any one of aminoacids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of SEQ ID NO: 1 and endsat any one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, or 135 of SEQ ID NO: 1. In some embodiments, the disclosureprovides for a method of treating, preventing, or reducing theprogression rate and/or severity of one or more complications ofpost-capillary pulmonary hypertension (e.g., WHO Group 2 and/or Group 5PH), comprising administering to a patient in need thereof an effectiveamount of a polypeptide comprising an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequencethat begins at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 of SEQ ID NO: 1 and ends at any one of amino acids 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, or 135 of SEQ ID NO: 1. Insome embodiments, the one or more complications of post-capillarypulmonary hypertension is selected from the group consisting of: smoothmuscle and/or endothelial cell proliferation in the pulmonary artery,angiogenesis in the pulmonary artery, dyspnea, chest pain, pulmonaryvascular remodeling, right ventricular hypertrophy, left ventricularhypertrophy, left atrium dilation, left ventricular fibrosis, rightventricular fibrosis, and pulmonary fibrosis. In some embodiments, thePcPH is isolated post-capillary pulmonary hypertension (IpcPH). In someembodiments, the PcPH is combined post- and pre-capillary PH (CpcPH).

In some embodiments, the patient has Group 2 pulmonary hypertension asrecognized by the World Health Organization (WHO). In some embodiments,the patient has pulmonary hypertension due to heart failure withpreserved left ventricular ejection fraction (LVEF). In someembodiments, the patient has pulmonary hypertension due to heart failurewith reduced left ventricular ejection fraction (LVEF). In someembodiments, the patient has valvular heart disease. In someembodiments, the patient has congenital/acquired cardiovascularconditions leading to post-capillary PH. In some embodiments, thepatient has Group 5 pulmonary hypertension as recognized by the WHO. Insome embodiments, the patient has pulmonary hypertension with unclearand/or multifactorial mechanisms. In some embodiments, the valvularheart disease is aortic regurgitation. In some embodiments, the valvularheart disease is aortic stenosis. In some embodiments, the valvularheart disease is mitral valve regurgitation. In some embodiments, thevalvular heart disease is mitral valve stenosis.

In some embodiments, the patient has a mean pulmonary arterial pressure(mPAP) selected from the group consisting of an mPAP of at least 20mmHg; an mPAP of at least 25 mmHg; an mPAP of at least 30 mmHg; an mPAPof at least 35 mmHg; an mPAP of at least 40 mmHg; an mPAP of at least 45mmHg; and an mPAP of at least 50 mmHg. In some embodiments, the methodreduces mPAP in the patient. In some embodiments, the method reduces themPAP in the patient by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%). In someembodiments, the method reduces the mPAP by at least 3 mmHg (e.g., atleast 3, 5, 7, 10, 12, 15, 20, or 25 mm Hg) in the patient.

In some embodiments, the patient has a pulmonary arterial wedge pressure(PAWP) of greater than 15 mmHg. In some embodiments, the methoddecreases the PAWP in the patient. In some embodiments, the methodreduces the PAWP in the patient by at least 10% (e.g., 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%).In some embodiments, the patient has a left ventricular end diastolicpressure (LVEDP) of greater than 15 mmHg. In some embodiments, themethod decreases the LVEDP in the patient. In some embodiments, themethod reduces the LVEDP in the patient by at least 10% (e.g., 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, thepatient has a diastolic pressure gradient (DPG) of less than 7 mmHg. Insome embodiments, the patient has a DPG of at least 7 mmHg. In someembodiments, the method decreases the DPG in the patient. In someembodiments, the method reduces the DPG in the patient by at least 10%(e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, or at least 80%). In some embodiments, the patient has atranspulmonary pressure gradient (TPG) of less than or equal to 12 mmHg. In some embodiments, the patient has a TPG of greater than 12 mm Hg.In some embodiments, the method decreases the TPG in the patient. Insome embodiments, the method reduces the TPG in the patient by at least10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, or at least 80%). In some embodiments, the patient has apulmonary vascular resistance (PVR) greater than or equal to 3 WoodUnits. In some embodiments, the method decreases the PVR in the patient.In some embodiments, the method reduces the PVR in the patient by atleast 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, or at least 80%).

In some embodiments, the method prevents the progression of IpcPH toCpcPH. In some embodiments, the method reduces the development of apre-capillary component of PH. In some embodiments, the patient haspreserved left ventricular ejection fraction. In some embodiments, thepreserved left ventricular ejection fraction is greater than 45%. Insome embodiments, the patient has reduced left ventricular ejectionfraction. In some embodiments, the reduced left ventricular fraction isless than 45%. In some embodiments, the preserved left ventricularfraction is measured using echocardiography. In some embodiments, thepatient has diastolic dysfunction of the left ventricle. In someembodiments, the patient has systolic dysfunction of the left ventricle.In some embodiments, the method decreases right ventricular hypertrophyin the patient. In some embodiments, the method decreases rightventricular hypertrophy in the patient by at least 10% (e.g., 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least80%). In some embodiments, the method decreases left ventricularhypertrophy in the patient. In some embodiments, the method decreasesleft ventricular hypertrophy in the patient by at least 10% (e.g., 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or atleast 80%). In some embodiments, the method decreases smooth musclehypertrophy in the patient. In some embodiments, the method decreasessmooth muscle hypertrophy in the patient by at least 10% (e.g., 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or atleast 80%). In some embodiments, the method decreases pulmonaryarteriole muscularity in the patient. In some embodiments, the methoddecreases pulmonary arteriole muscularity in the patient by at least 10%(e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, or at least 80%).

In some embodiments, the patient has a right ventricular systolicpressure (RVSP) of greater than 35 mmHg. In some embodiments, the methoddecreases the RVSP in the patient. In some embodiments, the methodreduces the RVSP in the patient by at least 10% (e.g., 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, thepatient has left ventricular fibrosis. In some embodiments, the methoddecreases the left ventricular fibrosis in the patient. In someembodiments, the method reduces the left ventricular fibrosis in thepatient by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,or at least 50%). In some embodiments, the patient has right ventricularfibrosis. In some embodiments, the method decreases the rightventricular fibrosis in the patient. In some embodiments, the methodreduces the right ventricular fibrosis in the patient by at least 10%(e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In someembodiments, the patient has pulmonary fibrosis. In some embodiments,the method decreases the pulmonary fibrosis in the patient. In someembodiments, the method reduces the pulmonary fibrosis in the patient byat least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least50%).

In some embodiments, the patient has a comorbidity selected from thegroup consisting of systemic hypertension, diabetes mellitus, obesity,coronary artery disease (CAD), heart failure, and anemia. In someembodiments, the method further comprises administering to the patientan additional active agent and/or supportive therapy. In someembodiments, the additional active agent and/or supportive therapy isselected from the group consisting of: beta-blockers,angiotensin-converting enzyme inhibitors (ACE inhibitors), angiotensinreceptor blockers (ARBs), neprilysin inhibitors, angiotensinreceptor-neprilysin inhibitors (ARNI), mineralocorticoid receptorantagonists (MRA), hyperpolarization-activated cyclic nucleotide-gated(HCN) channel blockers, diuretic agents, lipid-lowering medications,endothelin blockers, PDE5 inhibitors, prostacyclins, cardiacresynchronization therapy, valve replacement, valve repair, implantablecardioverter-defibrillator (ICD), or a left ventricular assist device(LVAD). In some embodiments, the additional active agent and/orsupportive therapy is selected from the group consisting of:prostacyclin and derivatives thereof (e.g., epoprostenol, treprostinil,and iloprost); prostacyclin receptor agonists (e.g., selexipag);endothelin receptor antagonists (e.g., thelin, ambrisentan, macitentan,and bosentan); calcium channel blockers (e.g., amlodipine, diltiazem,and nifedipine; anticoagulants (e.g., warfarin); diuretics; oxygentherapy; atrial septostomy; pulmonary thromboendarterectomy;phosphodiesterase type 5 inhibitors (e.g., sildenafil and tadalafil);activators of soluble guanylate cyclase (e.g., cinaciguat andriociguat); ASK-1 inhibitors (e.g., CIIA; SCH79797; GS-4997;MSC2032964A; 3H-naphtho[1,2,3-de]quiniline-2,7-diones, NQDI-1;2-thioxo-thiazolidines,5-bromo-3-(4-oxo-2-thioxo-thiazolidine-5-ylidene)-1,3-dihydro-indol-2-one);NF-κB antagonists (e.g., dh404, CDDO-epoxide; 2,2-difluoropropionamide;C28 imidazole (CDDO-Im); 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid(CDDO); 3-Acetyloleanolic Acid; 3-Triflouroacetyloleanolic Acid;28-Methyl-3-acetyloleanane; 28-Methyl-3-trifluoroacetyloleanane;28-Methyloxyoleanolic Acid; SZC014; SCZ015; SZC017; PEGylatedderivatives of oleanolic acid; 3-O-(beta-D-glucopyranosyl) oleanolicacid; 3-O-[beta-D-glucopyranosyl-(1→3)-beta-D-glucopyranosyl] oleanolicacid; 3-O-[beta-D-glucopyranosyl-(1→2)-beta-D-glucopyranosyl] oleanolicacid; 3-O-[beta-D-glucopyranosyl-(1→3)-beta-D-glucopyranosyl] oleanolicacid 28-O-beta-D-glucopyranosyl ester;3-O-[beta-D-glucopyranosyl-(1→2)-beta-D-glucopyranosyl] oleanolic acid28-O-beta-D-glucopyranosyl ester;3-O-[a-L-rhamnopyranosyl-(1→3)-beta-D-glucuronopyranosyl] oleanolicacid; 3-O-[alpha-L-rhamnopyranosyl-(1→3)-beta-D-glucuronopyranosyl]oleanolic acid 28-O-beta-D-glucopyranosyl ester;28-O-β-D-glucopyranosyl-oleanolic acid; 3-O-β-D-glucopyranosyl(1→3)-β-D-glucopyranosiduronic acid (CS1); oleanolic acid3-O-β-D-glucopyranosyl (1→3)-β-D-glucopyranosiduronic acid (CS2); methyl3,11-dioxoolean-12-en-28-olate (DIOXOL); ZCVI₄-2; Benzyl3-dehydr-oxy-1,2,5-oxadiazolo[3′,4′:2,3]oleanolate); eplerenone,spironolactone, ivabradine, implantable cardioverter-defibrillator(ICD), a left ventricular assist device (LVAD), or lung and/or hearttransplantation.

In some embodiments, the patient has elevated brain natriuretic peptide(BNP) levels as compared to a healthy patient. In some embodiments, thepatient has a BNP level of at least 100 pg/mL (e.g., 100, 150, 200, 300,400, 500, 1000, 3000, 5000, 10,000, 15,000, or 20,000 pg/mL). In someembodiments, the method decreases BNP levels in the patient by at least10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, or at least 80%). In some embodiments, the method decreasesBNP levels to normal levels (i.e., <100 pg/ml). In some embodiments, themethod decreases NT-proBNP levels in the patient. In some embodiments,the method decreases NT-proBNP levels in the patient by at least 10%(e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, or at least 80%). In some embodiments, the method decreasesNT-proBNP levels in the patient by at least 30%. In some embodiments,the method decreases NT-proBNP levels to normal levels. In someembodiments, the normal level of NT-proBNP is <100 pg/ml. In someembodiments, the method increases exercise capacity of the patient. Insome embodiments, the patient has a 6-minute walk distance from 150 to400 meters. In some embodiments, the patient has a 6-minute walkdistance from 150 to 550 meters. In some embodiments, the methodincreases the patient's 6-minute walk distance. In some embodiments, themethod increases the patient's 6-minute walk distance by at least 10meters (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125,150, 175, 200, 250, 300, or more than 400 meters). In some embodiments,the method reduces the patient's Borg dyspnea index (BDI). In someembodiments, the method reduces the patient's BDI by at least 0.5 indexpoints (e.g., at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 index points).

In some embodiments, the patient has decreased renal function. In someembodiments, the method further improves renal function. In someembodiments, the patient has Functional Class II or Class III pulmonaryhypertension in accordance with the World Health Organization'sfunctional classification system for pulmonary hypertension. In someembodiments, the patient has Functional Class I, Class II, Class III, orClass IV pulmonary hypertension as recognized by the World HealthOrganization. In some embodiments, the method prevents or delayspulmonary hypertension Functional Class progression (e.g., prevents ordelays progression from Functional Class I to Class II, Class II toClass III, or Class III to Class IV pulmonary hypertension as recognizedby the World Health Organization). In some embodiments, the methodpromotes or increases pulmonary hypertension Functional Class regression(e.g., promotes or increases regression from Class IV to Class III,Class III to Class II, or Class II to Class I pulmonary hypertension asrecognized by the World Health Organization). In some embodiments, thepatient has Functional Class II or Class III pulmonary hypertension inaccordance with the New York Heart Association's functionalclassification system for pulmonary hypertension. In some embodiments,the patient has Functional Class I, Class II, Class III, or Class IVpulmonary hypertension as recognized by the New York Heart Association.In some embodiments, the method prevents or delays pulmonaryhypertension Functional Class progression (e.g., prevents or delaysprogression from Functional Class I to Class II, Class II to Class III,or Class III to Class IV pulmonary hypertension as recognized by the NewYork Heart Association). In some embodiments, the method promotes orincreases pulmonary hypertension Functional Class regression (e.g.,promotes or increases regression from Class IV to Class III, Class IIIto Class II, or Class II to Class I pulmonary hypertension as recognizedby the New York Heart Association). In some embodiments, the methoddelays clinical worsening of PcPH. In some embodiments, the methoddelays clinical worsening of PcPH in accordance with the World HealthOrganization's functional classification system for pulmonaryhypertension. In some embodiments, the method delays clinical worseningof PcPH in accordance with the New York Heart Association's functionalclassification system for pulmonary hypertension. In some embodiments,the method reduces the risk of hospitalization for one or morecomplications associated with PcPH. In some embodiments, the patient hasa hemoglobin level from >8 and <15 g/dl.

In some embodiments, the patient has been treated with one or morevasodilators. In some embodiments, the patient has been treated with oneor more agents selected from the group consisting of: phosphodiesterasetype 5 inhibitors, soluble guanylate cyclase stimulators, prostacyclinreceptor agonist, and endothelin receptor antagonists. In someembodiments, the one or more agents is selected from the groupconsisting of: bosentan, sildenafil, beraprost, macitentan, selexipag,epoprostenol, treprostinil, iloprost, ambrisentan, and tadalafil. Insome embodiments, the method further comprises administration of one ormore vasodilators. In some embodiments, the method further comprisesadministration of one or more agents selected from the group consistingof: phosphodiesterase type 5 inhibitors, soluble guanylate cyclasestimulators, prostacyclin receptor agonist, and endothelin receptorantagonists. In some embodiments, the one or more agents is selectedfrom the group consisting of: bosentan, sildenafil, beraprost,macitentan, selexipag, epoprostenol, treprostinil, iloprost,ambrisentan, and tadalafil.

In some embodiments, the ActRII polypeptide comprises an amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence of amino acids corresponding to residues 30-110 of SEQ IDNO: 1. In some embodiments, the ActRII polypeptide comprises an aminoacid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical tothe amino acid sequence SEQ ID NO: 2. In some embodiments, the ActRIIpolypeptide comprises an amino acid sequence that is at least 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3.In some embodiments, the ActRII polypeptide is a fusion protein furthercomprising an Fc domain of an immunoglobulin. In some embodiments, theFc domain of the immunoglobulin is an Fc domain of an IgG1immunoglobulin. In some embodiments, the Fc fusion protein furthercomprises a linker domain positioned between the ActRII polypeptidedomain and the Fc domain of the immunoglobulin. In some embodiments, thelinker domain is selected from the group consisting of: TGGG (SEQ ID NO:20), TGGGG (SEQ ID NO: 18), SGGGG (SEQ ID NO: 22), GGGGS (SEQ ID NO:22), GGG (SEQ ID NO: 16), GGGG (SEQ ID NO: 17), and SGGG (SEQ ID NO:21). In some embodiments, the ActRII polypeptide comprises an amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 23. In some embodiments, thepolypeptide comprises an amino acid sequence that is at least 90%identical to an amino acid sequence corresponding to residues 30-110 ofSEQ ID NO: 1, wherein the polypeptide binds to activing and/or GDF11. Insome embodiments, the polypeptide comprises an amino acid sequence thatis at least 90% identical to an amino acid sequence corresponding toresidues 21-135 of SEQ ID NO: 1, wherein the polypeptide binds toactiving and/or GDF11. In some embodiments, the polypeptide islyophilized. In some embodiments, the polypeptide is soluble. In someembodiments, the polypeptide is administered using subcutaneousinjection. In some embodiments, the polypeptide is administered every 4weeks. In some embodiments, the polypeptide is part of a homodimerprotein complex. In some embodiments, the polypeptide is glycosylated.In some embodiments, the polypeptide has a glycosylation patternobtainable by expression in a Chinese hamster ovary cell. In someembodiments, the ActRII polypeptide binds to one or more ligandsselected from the group consisting of: activin A, activin B, and GDF11.In some embodiments, the ActRII polypeptide further binds to one or moreligands selected from the group consisting of: BMP10, GDF8, and BMP6.

In certain aspects, the disclosure relates to a kit comprising alyophilized polypeptide and an injection device, wherein the polypeptideis an ActRII polypeptide comprising an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequencethat begins at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 of SEQ ID NO: 1 and ends at any one of amino acids 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, or 135 of SEQ ID NO: 1. Insome embodiments, the polypeptide is a polypeptide comprising an aminoacid sequence that is at least 90% identical to an amino acid sequencecorresponding to residues 30-110 of SEQ ID NO: 1. In some embodiments,the polypeptide is a polypeptide comprising an amino acid sequence thatis at least 95% identical to the amino acid sequence corresponding toresidues 30-110 of SEQ ID NO: 1. In some embodiments, the polypeptide isa polypeptide comprising an amino acid sequence that is at least 99%identical to the amino acid sequence corresponding to residues 30-110 ofSEQ ID NO: 1. In some embodiments, the polypeptide is a polypeptidecomprising the amino acid sequence corresponding to residues 30-110 ofSEQ ID NO: 1. In some embodiments, the polypeptide is a polypeptideconsisting of the amino acid sequence corresponding to residues 30-110of SEQ ID NO: 1. In some embodiments, the polypeptide is a polypeptidecomprising an amino acid sequence that is at least 90% identical to theamino acid sequence corresponding to residues 21-135 of SEQ ID NO: 1. Insome embodiments, the polypeptide is a polypeptide comprising an aminoacid sequence that is at least 95% identical to the amino acid sequencecorresponding to residues 21-135 of SEQ ID NO: 1.

In some embodiments, the polypeptide is a polypeptide comprising anamino acid sequence that is at least 99% identical to the amino acidsequence corresponding to residues 21-135 of SEQ ID NO: 1. In someembodiments, the polypeptide is a polypeptide comprising the amino acidsequence corresponding to residues 21-135 of SEQ ID NO: 1. In someembodiments, the polypeptide is a polypeptide consisting of the aminoacid sequence corresponding to residues 21-135 of SEQ ID NO: 1. In someembodiments, the polypeptide is a polypeptide comprising an amino acidsequence that is at least 90% identical to the amino acid sequence ofSEQ ID NO: 2. In some embodiments, the polypeptide is a polypeptidecomprising an amino acid sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO: 2. In some embodiments, thepolypeptide is a polypeptide comprising an amino acid sequence that isat least 99% identical to the amino acid sequence of SEQ ID NO: 2. Insome embodiments, the polypeptide is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 2. In some embodiments, the polypeptide is apolypeptide consisting of the amino acid sequence of SEQ ID NO: 2. Insome embodiments, the polypeptide is a polypeptide comprising an aminoacid sequence that is at least 90% identical to the amino acid sequenceof SEQ ID NO: 3. In some embodiments, the polypeptide is a polypeptidecomprising an amino acid sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO: 3. In some embodiments, thepolypeptide is a polypeptide comprising an amino acid sequence that isat least 99% identical to the amino acid sequence of SEQ ID NO: 3. Insome embodiments, the polypeptide is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 3. In some embodiments, the polypeptide is apolypeptide consisting of the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the polypeptide is a fusion protein furthercomprising an Fc domain of an immunoglobulin. In some embodiments, theimmunoglobulin is an Fc domain of an IgG1 immunoglobulin. In someembodiments, the fusion protein further comprises a linker domainpositioned between the polypeptide domain and the Fc domain of theimmunoglobulin. In some embodiments, the linker domain is selected fromthe group consisting of: TGGG (SEQ ID NO: 20), TGGGG (SEQ ID NO: 18),SGGGG (SEQ ID NO: 19), GGGGS (SEQ ID NO: 22), GGG (SEQ ID NO: 16), GGGG(SEQ ID NO: 17), and SGGG (SEQ ID NO: 21). In some embodiments, thelinker domain comprises TGGG (SEQ ID NO: 20). In some embodiments, theActRII polypeptide comprises an amino acid sequence that is at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQID NO: 23. In some embodiments, the ActRII polypeptide comprises theamino acid sequence of SEQ ID NO: 23. In some embodiments, the ActRIIpolypeptide consists of the amino acid sequence of SEQ ID NO: 23. Insome embodiments, the polypeptide is part of a homodimer proteincomplex. In some embodiments, the polypeptide is glycosylated. In someembodiments, the polypeptide binds to one or more ligands selected fromthe group consisting of: activin A, activin B, and GDF11. In someembodiments, the polypeptide further binds to one or more ligandsselected from the group consisting of: BMP10, GDF8, and BMP6. In someembodiments, the polypeptide binds to activin and/or GDF11.

In some embodiments, the kit comprises one or more vials containing thelyophilized polypeptide. In some embodiments, the injection devicecomprises a pre-filled syringe. In some embodiments, the injectiondevice comprises a pump apparatus. In some embodiments, the pumpapparatus comprises an electromechanical pumping assembly. In someembodiments, the pump apparatus is a wearable pump apparatus. In someembodiments, the pre-filled syringe comprises a reconstitution solution.In some embodiments, the reconstitution solution comprises apharmaceutically acceptable carrier and/or excipient. In someembodiments, the pharmaceutically acceptable carrier is selected fromsaline solution, purified water, or sterile water for injection. In someembodiments, the pharmaceutically acceptable excipient is selected froma buffering agent [e.g., citric acid (monohydrate) and/or trisodiumcitrate (dehydrate)], a surfactant (e.g., polysorbate 80), a stabilizer(e.g., sucrose), and a lyoprotectant (e.g., sucrose).

In some embodiments, the injection device comprises a vial adapter. Insome embodiments, the vial adapter is capable of attaching to a vial. Insome embodiments, the vial adapter is capable of attaching to apre-filled syringe. In some embodiments, the pre-filled syringe and thevial are attached to opposite ends of the vial adapter. In someembodiments, the reconstitution solution is transferred from thepre-filled syringe to the vial. In some embodiments, the lyophilizedpolypeptide is reconstituted into a sterile injectable solution. In someembodiments, the lyophilized polypeptide is reconstituted into a sterileinjectable solution prior to use. In some embodiments, the sterileinjectable solution is sterile water for injection.

In some embodiments, the sterile injectable solution is administeredparenterally. In some embodiments, the injection device is used toadminister the sterile injectable solution parenterally. In someembodiments, the sterile injectable solution is administered viasubcutaneous injection. In some embodiments, the sterile injectablesolution is administered via intradermal injection. In some embodiments,the sterile injectable solution is administered via intramuscularinjection. In some embodiments, the sterile injectable solution isadministered via intravenous injection. In some embodiments, the sterileinjectable solution is self-administered. In some embodiments, thesterile injectable solution comprises a therapeutically effective dose.In some embodiments, the therapeutically effective dose comprises aweight based dose. In some embodiments, the lyophilized polypeptide isadministered every 4 weeks.

In some embodiments, the kit is used to treat post-capillary pulmonaryhypertension (PcPH). In some embodiments, the PcPH is isolatedpost-capillary pulmonary hypertension (IpcPH). In some embodiments, thePcPH is combined post- and pre-capillary PH (CpcPH). In someembodiments, the patient has Group 2 pulmonary hypertension asrecognized by the WHO. In some embodiments, the patient has pulmonaryhypertension due to heart failure with preserved left ventricularejection fraction (LVEF). In some embodiments, the patient has pulmonaryhypertension due to heart failure with reduced left ventricular ejectionfraction (LVEF). In some embodiments, the patient has valvular heartdisease. In some embodiments, the patient has congenital/acquiredcardiovascular conditions leading to post-capillary PH. In someembodiments, the patient has Group 5 pulmonary hypertension asrecognized by the WHO. In some embodiments, the patient has pulmonaryhypertension with unclear and/or multifactorial mechanisms. In someembodiments, the shelf life of the lyophilized polypeptide is at least1, 1.5, 2, 2.5, or 3 years. In some embodiments, the lyophilizedpolypeptide is reconstituted. In some embodiments, the reconstitutedpolypeptide has a shelf life of at least 2 hrs, 3 hrs, or 4 hrs.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing/photographexecuted in color. Copies of this patent with colordrawing(s)/photograph(s) will be provided by the Office upon request andpayment of the necessary fee.

FIG. 1 shows an alignment of extracellular domains of human ActRIIB (SEQID NO: 31) and human ActRIIA (SEQ ID NO: 2) with the residues that arededuced herein, based on composite analysis of multiple ActRIIB andActRIIA crystal structures, to directly contact ligand indicated withboxes.

FIG. 2 shows a multiple sequence alignment of various vertebrate ActRIIAproteins and human ActRIIA (SEQ ID NOs: 6-10 and 36-38).

FIG. 3 shows multiple sequence alignment of Fc domains from human IgGisotypes using Clustal 2.1. Hinge regions are indicated by dottedunderline. Double underline indicates examples of positions engineeredin IgG1 Fc (SEQ ID NO: 32) to promote asymmetric chain pairing and thecorresponding positions with respect to other isotypes IgG2 (SEQ ID NO:33), IgG3 (SEQ ID NO: 34) and IgG4 (SEQ ID NO: 35).

FIGS. 4A and 4B show the purification ofActRIIA-hFc expressed in CHOcells. The protein purifies as a single, well-defined peak as visualizedby sizing column (FIG. 4A) and Coomassie stained SDS-PAGE (FIG. 4B)(left lane: molecular weight standards; right lane: ActRIIA-hFc).

FIGS. 5A and 5B show the binding of ActRIIA-hFc to activin (FIG. 5A) andGDF-11 (FIG. 5B), as measured by Biacore™ assay.

FIG. 6 shows a schematic image of a linearized version ofcardiopulmonary circulation and the regions associated with varioustypes of PH. The difference between pre-capillary pulmonaryhypertension, isolated post-capillary pulmonary hypertension, andcombined post- and pre-capillary pulmonary hypertension are based onpulmonary hemodynamic parameters and the involvement of various regionsof the cardiopulmonary system (pre and/or post capillary regions).Abbreviations are as follows: VC—vena cava; RA—right atrium; RV—rightventricle; PA—pulmonary artery; PC—pulmonary capillaries; PV—pulmonaryventricles; LA—left atrium; LV—left ventricle; AO—Aorta. See, e.g., ArasM A, et al. Curr Cardiol Rep. 2019; 21(7):62 and Galib N. et al. EurHeart J. 2018; 39(15):1265-1268.

FIG. 7 shows a schematic image of a linearized version ofcardiopulmonary circulation and the hemodynamic parameters associatedwith pre-capillary PH. Abbreviations are as follows: VC—vena cava;RA—right atrium; RV—right ventricle; PA—pulmonary artery; PC—pulmonarycapillaries; PV—pulmonary ventricles; LA—left atrium; LV—left ventricle;AO—Aorta; mPAP—mean pulmonary arterial pressure; PAWP—pulmonary arterialwedge pressure; PVR—pulmonary vascular resistance. Id.

FIG. 8 shows a schematic image of a linearized version ofcardiopulmonary circulation and the hemodynamic parameters associatedwith isolated post-capillary PH (IpcPH). Abbreviations are as follows:VC—vena cava; RA—right atrium; RV—right ventricle; PA—pulmonary artery;PC—pulmonary capillaries; PV—pulmonary ventricles; LA—left atrium;LV—left ventricle; AO—Aorta; mPAP—mean pulmonary arterial pressure;PAWP—pulmonary arterial wedge pressure; PVR—pulmonary vascularresistance. Id.

FIG. 9 shows a schematic image of a linearized version ofcardiopulmonary circulation and the hemodynamic parameters associatedwith combined post- and pre-capillary PH (CpcPH). Abbreviations are asfollows: VC—vena cava; RA—right atrium; RV—right ventricle; PA—pulmonaryartery; PC—pulmonary capillaries; PV—pulmonary ventricles; LA—leftatrium; LV—left ventricle; AO—Aorta; mPAP—mean pulmonary arterialpressure; PAWP—pulmonary arterial wedge pressure; PVR—pulmonary vascularresistance. Id.

FIGS. 10-14 shows the therapeutic effect of ActRIIA-mFc in a TAC-PHmodel based on endpoints for left ventricle function. Twenty-six C57/B6male mice (10 wks old) underwent TAC pulmonary hypertension surgery(TAC-PH) and ten age-matched animals underwent a mock surgical procedure(Sham) at day 0. Two weeks after the surgery, TAC-PH mice wererandomized into two groups. i) fourteen mice were injectedsubcutaneously with vehicle control (phosphate buffered saline (PBS)),twice weekly for 4 weeks starting from day 14 after surgery,“TAC-PH/PBS”; and a ii) twelve mice were injected subcutaneously withActRIIA-mFc at a dose of 10 mg/kg twice weekly for 4 weeks starting fromday 14 after TAC surgery, “TAC-PH/ActRIIA-mFc”. FIGS. 10-14 showendpoints for left ventricle function, including changes in cardiachypertrophy heart weight/body weight (HW/BW) (FIG. 10 ), LV functionparameters fractional shorting (FIG. 11 ) and LV ejection fraction (FIG.12 ); and LV diastolic function parameters E/E′ [Ratio of mitrial inflowvelocity (E) to mitrial annular velocity (E′)] (FIG. 13 ) andisovolumetric relaxation time (IVRT) (FIG. 14 ). Relative to“TAC-PH/PBS” treated mice, “TAC-PH/ActRIIA-mFc” treated micedemonstrated a significant effect of ActRIIA-mFc in reducing cardiachypertrophy and improving cardiac function. Statistical significance (pvalue) is depicted as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001for comparison between “Sham” and sample “TAC-PH/PBS”. Statisticalsignificance (p value) is depicted as #p<0.05, ##p<0.01, ###p<0.001, and####p<0.0001 for comparison between “Sham” and sample“TAC-PH/ActRIIA-mFc”. Statistical significance (p value) is depicted as@ p<0.05, @@p<0.01, @@@p<0.001, and @@@@p<0.0001 for comparison betweensample “TAC-PH/PBS” and sample “TAC-PH/ActRIIA-mFc”.

FIGS. 15-18 show the therapeutic effect of ActRIIA-mFc in a TAC-PH modelbased on endpoints for right ventricle function. Twenty-six C57/B6 malemice (10 wks old) underwent TAC pulmonary hypertension surgery (TAC-PH)and ten age-matched animals underwent a mock surgical procedure (Sham)at day 0. Two weeks after the surgery, TAC-PH mice were randomized intotwo groups. i) fourteen mice were injected subcutaneously with vehiclecontrol (phosphate buffered saline (PBS)), twice weekly for 4 weeksstarting from day 14 after surgery, “TAC-PH/PBS”; and a ii) twelve micewere injected subcutaneously with ActRIIA-mFc at a dose of 10 mg/kgtwice weekly for 4 weeks starting from day 14 after TAC surgery,“TAC-PH/ActRIIA-mFc”. FIGS. 15-18 show endpoints for right ventriclefunction, including RV remodeling parameter right ventricular free wallthickness (RVFWT) (FIG. 15 ), RV remodeling and function parametertricuspid annular plane systolic excursion (TAPSE) (FIG. 16 ), and RVfunction parameters RV stroke work (FIG. 17 ) and RV contractility(dP/dT) (FIG. 18 ). Relative to “TAC-PH/PBS” treated mice,“TAC-PH/ActRIIA-mFc” treated mice demonstrated a significant effect ofActRIIA-mFc in improving right heart remodeling and function.Statistical significance (p value) is depicted as *p<0.05, **p<0.01,***p<0.001, and ****p<0.0001 for comparison between “Sham” and sample“TAC-PH/PBS”. Statistical significance (p value) is depicted as #p<0.05,##p<0.01, ###p<0.001, and ####p<0.0001 for comparison between “Sham” andsample “TAC-PH/ActRIIA-mFc”. Statistical significance (p value) isdepicted as @ p<0.05, @@p<0.01, @@@p<0.001, and @@@@p<0.0001 forcomparison between sample “TAC-PH/PBS” and sample “TAC-PH/ActRIIA-mFc”.

FIGS. 19 and 20 show the therapeutic effect of ActRIIA-mFc in a TAC-PHmodel based on endpoints for lung remodeling. Twenty-six C57/B6 malemice (10 wks old) underwent TAC pulmonary hypertension surgery (TAC-PH)and ten age-matched animals underwent a mock surgical procedure (Sham)at day 0. Two weeks after the surgery, TAC-PH mice were randomized intotwo groups. i) fourteen mice were injected subcutaneously with vehiclecontrol (phosphate buffered saline (PBS)), twice weekly for 4 weeksstarting from day 14 after surgery, “TAC-PH/PBS”; and a ii) twelve micewere injected subcutaneously with ActRIIA-mFc at a dose of 10 mg/kgtwice weekly for 4 weeks starting from day 14 after TAC surgery,“TAC-PH/ActRIIA-mFc”. FIGS. 19 and 20 show endpoints for lungremodeling, including ratio of lung weight to tibia length (LW/TL) (FIG.19 ) and lung fibrosis percentage (FIG. 20 ). Relative to “TAC-PH/PBS”treated mice, “TAC-PH/ActRIIA-mFc” treated mice demonstrated asignificant effect of ActRIIA-mFc in reducing pulmonary remodeling andfibrosis. Statistical significance (p value) is depicted as *p<0.05,**p<0.01, ***p<0.001, and ****p<0.0001 for comparison between “Sham” andsample “TAC-PH/PBS”. Statistical significance (p value) is depicted as#p<0.05, ##p<0.01, ###p<0.001, and ####p<0.0001 for comparison between“Sham” and sample “TAC-PH/ActRIIA-mFc”. Statistical significance (pvalue) is depicted as @ p<0.05, @@p<0.01, @@@p<0.001, and @@@@p<0.0001for comparison between sample “TAC-PH/PBS” and sample“TAC-PH/ActRIIA-mFc”.

FIG. 21 shows components of a kit comprising a lyophilized polypeptideand an injection device. A vial (1) holds lyophilized polypeptide,reconstituted sterile injectable solution, or sterile injectablesolution. A prefilled syringe (2) containing a reconstitution solutionis used to reconstitute lyophilized polypeptide from (1) into a sterileinjectable solution. A vial adapter (3) couples the vial (1) to thepre-filled syringe (2) via attachment to the vial at one end, andattachment to the pre-filled syringe at an opposite end. A syringe (4)and needle (5) are provided for administration of sterile injectablesolution. Swab wipes (6) are provided for sterilization of individualkit components.

FIGS. 22-25 show that treatment with an ActRIIA-mFc fusion proteinimproves diastolic dysfunction in a rat model of left ventriculardiastolic dysfunction (also referred to as HEpEF) group 2 (subgroup 2.2)pulmonary hypertension (PH). The experimental strategy used to test thepreventative effects of ActRIIA-mFc in the rat model of HEpEF is shownin FIG. 22 . FIGS. 23-25 show endpoints for left ventricular function,including the left ventricular ejection fraction (FIG. 23 ); LVdiastolic function parameters E/E′ [Ratio of mitrial inflow velocity (E)to mitrial annular velocity (E′)] (FIG. 24 ); and isovolumetricrelaxation time (IVRT) (FIG. 25 ). Statistical significance (p value) isdepicted as *p<0.05, **p<0.01, and ***p<0.001.

FIGS. 26-28 show that treatment with an ActRIIA-mFc fusion proteinreduces left heart remodeling in a rat model of left ventriculardiastolic dysfunction (also referred to as HEpEF) group 2 (subgroup 2.2)pulmonary hypertension (PH). FIGS. 26-28 show endpoints for left heartremodeling, including changes in ratio of heart weight to tibia length(HW/TL) (FIG. 26 ); interventricular septal dimension at diastole (IVSd)(FIG. 27 ); and left ventricular mass (LVM) (FIG. 28 ). Statisticalsignificance (p value) is depicted as *p<0.05, **p<0.01, and ***p<0.001.

FIGS. 29-31 show that treatment with an ActRIIA-mFc fusion proteinreduces right ventricular systolic pressure (RVSP) and improves rightventricular function in a rat model of left ventricular diastolicdysfunction (also referred to as HEpEF) group 2 (subgroup 2.2) pulmonaryhypertension (PH). FIGS. 29-31 show endpoints for right ventricularfunction, including changes in right ventricular free wall thickness(FIG. 29 ); pulmonary artery acceleration time (PAAT) (FIG. 30 ); andright ventricular systolic pressure (RVSP) (FIG. 31 ). Statisticalsignificance (p value) is depicted as *p<0.05 and **p<0.01.

FIGS. 32-34 show that treatment with an ActRIIA-mFc fusion proteinsignificantly reduced the fibrosis in LV, RV and lung in a rat model ofleft ventricular diastolic dysfunction (also referred to as HEpEF) group2 (subgroup 2.2) pulmonary hypertension (PH). FIGS. 32-34 show areduction in fibrosis, including changes in left ventricular fibrosis(FIG. 32 ); right ventricular fibrosis (FIG. 33 ); and lung fibrosis(FIG. 34 ). Statistical significance (p value) is depicted as *p<0.05and **p<0.01.

FIGS. 35-38 show that treatment with an ActRIIA-mFc fusion proteinsignificantly improves hyperglycemia and glucose intolerance in a ratmodel of left ventricular diastolic dysfunction (also referred to asHEpEF) group 2 (subgroup 2.2) pulmonary hypertension (PH).

FIGS. 35-38 show endpoints for hyperglycemia and glucose intolerance,including changes in body weight (FIG. 35 ); fasting glucose (FIG. 36 );blood glucose (FIG. 37 ); and glucose/creatine ratio (FIG. 38 ).Statistical significance (p value) is depicted as *p<0.05, **p<0.01, and***p<0.001.

FIGS. 39-43 show that treatment with an ActRIIA-mFc fusion proteininhibits cardiac remodeling and improves LV function in a mouse model ofPH due to heart failure with reduced LVEF (also referred to as HErEF)group 2 (subgroup 2.1) pulmonary hypertension (PH) and valvular heartdisease (subgroup 2.3). The experimental strategy used to test thepreventative effects of ActRIIA-mFc in the rat model of HErEF is shownin FIG. 39 . FIGS. 40-43 show endpoints for left ventricle function,including changes in cardiac hypertrophy heart weight/tibia length(HW/TL) (FIG. 41 ), LV function parameters such as LV ejection fraction(FIG. 40 ), LV diastolic function parameters E/E′ [Ratio of mitralinflow velocity (E) to mitral annular velocity (E′)] (FIG. 42 ) andisovolumetric relaxation time (IVRT) (FIG. 43 ). Relative to “TAC PBS”treated mice, “TAC ActRIIA-mFc” treated mice demonstrated a significanteffect of ActRIIA-mFc in inhibiting cardiac remodeling and improving LVfunction. Statistical significance (p value) is depicted as *p<0.05,**p<0.01, ***p<0.001, and ****p<0.0001 for comparison between “TAC PBS”and sample “TAC ActRIIA-mFc”. Statistical significance (p value) isdepicted as #p<0.05, ##p<0.01, ###p<0.001, and ####p<0.0001 forcomparison between “Sham” and sample “TAC PBS.”

FIGS. 44-46 show the therapeutic effect of ActRIIA-mFc in a TAC-PH modelbased on endpoints for right ventricle function. FIGS. 44-46 showendpoints for right ventricle function including right ventricularsystolic pressure (RVSP) (FIG. 44 ), right ventricular free wallthickness (RVFWT) (FIG. 45 ), and pulmonary artery acceleration time(PAAT) (FIG. 46 ). Relative to “TAC PBS” treated mice, “TAC ActRIIA-mFc”mice treated with either 3 mpk and 10 mpk demonstrated a significanteffect of ActRIIA-mFc in reducing RVSP and improving RV function.Statistical significance (p value) is depicted as *p<0.05, **p<0.01,***p<0.001, and ****p<0.0001 for comparison between “TAC PBS” and sample“TAC ActRIIA-mFc.” Statistical significance (p value) is depicted as#p<0.05, ##p<0.01, ###p<0.001, and ####p<0.0001 for comparison between“Sham” and sample “TAC PBS.”

FIGS. 47-49 show the therapeutic effect of ActRIIA-mFc in a TAC-PH modelbased on endpoints for fibrosis in the left ventricle (LV), rightventricle (RV), and lung. FIGS. 47-49 show endpoints for fibrosis in theleft ventricle (LV) (FIG. 47 ), right ventricle (RV) (FIG. 48 ), andlung (FIG. 49 ). Relative to “TAC PBS” treated mice, “TAC ActRIIA-mFc”mice treated with either 3 mpk or 10 mpk demonstrated a significanteffect of ActRIIA-mFc in reducing fibrosis in the LV (FIG. 47 ), RV(FIG. 48 ), and lung (FIG. 49 ). Statistical significance (p value) isdepicted as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 forcomparison between “TAC PBS” and sample “TAC ActRIIA-mFc.” Statisticalsignificance (p value) is depicted as #p<0.05, ##p<0.01, ###p<0.001, and####p<0.0001 for comparison between “Sham” and sample “TAC PBS.”

FIGS. 50-55 show that treatment with an ActRIIA-mFc fusion proteinreduces right ventricular systolic pressure (RVSP) and improvescardiopulmonary function in a rat model of left ventricular diastolicdysfunction (also referred to as HEpEF) group 2 (subgroup 2.2) pulmonaryhypertension (PH). The experimental strategy used to test thepreventative effects of an ActRIIA-mFc fusion protein in the rat modelof HEpEF is shown in FIG. 50 . FIGS. 51-55 show endpoints for rightventricular function, including changes in pulmonary artery accelerationtime (PAAT) (FIG. 51 ); right ventricular systolic pressure (RVSP) (FIG.52 ); right ventricular wall thickness (RVWT) (FIG. 53 ); tricuspidannular plane systolic excursion (TAPSE) (FIG. 54 ); and Fulton index,calculated as the ratio of right ventricular weight (RV) to weight ofthe combined left ventricle and septum (LV+S) (FIG. 55 ). Statisticalsignificance (p value) is depicted as *p<0.05, **p<0.01, ***p<0.001, and****p<0.0001.

DETAILED DESCRIPTION 1. Overview

The present disclosure relates to compositions and methods of treatingpost-capillary pulmonary hypertension (e.g., WHO Group 2 and/or Group 5PH) comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide as described herein. In certainembodiments, the present disclosure provides methods of treating orpreventing post-capillary pulmonary hypertension (PcPH) in an individualin need thereof through administering to the individual atherapeutically effective amount of an ActRII polypeptide as describedherein. In certain embodiments, the present disclosure provides methodsof treating or preventing combined post- and pre-capillary PH in anindividual in need thereof through administering to the individual atherapeutically effective amount of an ActRII polypeptide as describedherein.

Pulmonary hypertension due to left heart disease (PH-LHD) (also known asWHO Group 2 PH) is a complex pathophenotype that, when present, mayresult in an increased susceptibility to adverse events and a worseclinical outcome. Among those patients with PH-LHD, two phenotypes havebeen described: 1) a group of isolated post-capillary (IpcPH) or“passive” PH in which elevated pulmonary pressures are reversible and inproportion to increases in left atrial pressure, and 2) a group with“pre-capillary” component [combined post-capillary and pre-capillary PH(CpcPH)] whose pulmonary hypertension is worse than can be fullyexplained by passive elevation secondary to elevated left atrialpressure. This latter group, CpcPH, may have comorbid pulmonary vascularremodeling and therefore may demonstrate persistent PH afterinterventions to lower left sided filling pressures.

PH-LHD is sometimes defined as patients having a pulmonary capillarywedge pressure (PCWP)>15 mmHg and a mean pulmonary artery pressure(mPAP)≥25 mmHg (or a mean pulmonary artery pressure (mPAP)≥20 mmHg underupdated guidelines). PH-LHD occurs as a consequence of the backwardtransmission of high left sided filling pressures, mainly driven by LVdiastolic function, directly to the post-capillary pulmonary vesselsand, thereby, to the rest of the pulmonary circulation. In someembodiments, PH-LHD is driven by both systolic and diastolicdysfunction. PH-LHD may be associated with or caused by PH due to heartfailure with preserved left ventricle ejection fraction (LVEF) [alsoknown as HFpEF], PH due to heart failure with reduced LVEF (also knownas HFrEF), valvular heart disease, or congenital/acquired cardiovascularconditions leading to post-capillary PH. Compared with PAH, patientswith PH-LHD are often older, female, with a higher prevalence ofcardiovascular co-morbidities and most, if not all, of the features ofmetabolic syndrome.

For WHO Group 2 (PH-LHD) and Group 5 PH patients, there are no approvedspecific therapies available beyond treatment of the underlying disease.Most PH-LHD therapies target the underlying condition (e.g., repair ofvalvular heart disease) rather than specifically treating PH. The lackof specific therapies is particularly problematic because PH-LHD is themost common cause of PH in western countries and its presence commonlyresults in adverse course of the disease. Specifically, the presence ofPH-LHD can result in more severe symptoms in LHD, worse exercisetolerance, and a negative impact on outcome. Accordingly, there is ahigh unmet need for new treatments for post-capillary pulmonaryhypertension (e.g., WHO Group 2 and/or Group 5 PH) and these treatmentswould have the potential to positively affect large numbers of patients.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this disclosure and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification to provide additional guidanceto the practitioner in describing the compositions and methods of thedisclosure and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which it isused.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

“Percent (%) sequence identity” with respect to a reference polypeptide(or nucleotide) sequence is defined as the percentage of amino acidresidues (or nucleic acids) in a candidate sequence that are identicalto the amino acid residues (or nucleic acids) in the referencepolypeptide (nucleotide) sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid (nucleic acid) sequenceidentity values are generated using the sequence comparison computerprogram ALIGN-2. The ALIGN-2 sequence comparison computer program wasauthored by Genentech, Inc., and the source code has been filed withuser documentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available from Genentech, Inc., SouthSan Francisco, Calif., or may be compiled from the source code. TheALIGN-2 program should be compiled for use on a UNIX operating system,including digital UNIX V4.0D. All sequence comparison parameters are setby the ALIGN-2 program and do not vary.

“Agonize”, in all its grammatical forms, refers to the process ofactivating a protein and/or gene (e.g., by activating or amplifying thatprotein's gene expression or by inducing an inactive protein to enter anactive state) or increasing a protein's and/or gene's activity.

“Antagonize”, in all its grammatical forms, refers to the process ofinhibiting a protein and/or gene (e.g., by inhibiting or decreasing thatprotein's gene expression or by inducing an active protein to enter aninactive state) or decreasing a protein's and/or gene's activity.

The terms “about” and “approximately” as used in connection with anumerical value throughout the specification and the claims denotes aninterval of accuracy, familiar and acceptable to a person skilled in theart. In general, such interval of accuracy is ±10%. Alternatively, andparticularly in biological systems, the terms “about” and“approximately” may mean values that are within an order of magnitude,preferably ≤5-fold and more preferably ≤2-fold of a given value.

Numeric ranges disclosed herein are inclusive of the numbers definingthe ranges.

The terms “a” and “an” include plural referents unless the context inwhich the term is used clearly dictates otherwise. The terms “a” (or“an”), as well as the terms “one or more,” and “at least one” can beused interchangeably herein. Furthermore, “and/or” where used herein isto be taken as specific disclosure of each of the two or more specifiedfeatures or components with or without the other. Thus, the term“and/or” as used in a phrase such as “A and/or B” herein is intended toinclude “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, theterm “and/or” as used in a phrase such as “A, B, and/or C” is intendedto encompass each of the following aspects: A, B, and C; A, B, or C; Aor C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone);and C (alone).

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or groups of integers but not the exclusion of anyother integer or group of integers.

2. ActRII Polypeptides

In certain aspects, the disclosure relates to ActRII polypeptides anduses thereof (e.g., of treating, preventing, or reducing the progressionrate and/or severity of post-capillary pulmonary hypertension (PcPH) orone or more complications of PcPH). As used herein, the term “ActRII”refers to the family of type II activin receptors. This family includesactivin receptor type IIA (ActRIIA) and activin receptor type IIB(ActRIIB).

In certain embodiments, the present disclosure relates to ActRIIpolypeptides having an amino acid sequence that is at least 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to an amino acid sequence as set forth inanyone of SEQ ID NOs: 1, 2, 3, 23, 27, 30, and 41. In other embodiments,the present disclosure relates to ActRII polypeptides having an aminoacid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical toan amino acid sequence as set forth in anyone of SEQ ID NOs: 31, 39, and40. As used herein, the term “ActRII” refers to a family of activinreceptor type IIA (ActRIIA) proteins, a family of activin receptor typeIIB (ActRIIB) proteins, or combinations and/or variants thereof. TheActRII polypeptides can be derived from any species and include variantsderived from such ActRII proteins by mutagenesis or other modification.Reference to ActRII herein is understood to be a reference to any one ofthe currently identified forms. Members of the ActRII family aregenerally transmembrane proteins, composed of a ligand-bindingextracellular domain comprising a cysteine-rich region, a transmembranedomain, and a cytoplasmic domain with predicted serine/threonine kinaseactivity.

The term ActRII polypeptide includes polypeptides comprising anynaturally occurring polypeptide of an ActRII family member as well asany variants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Examples of suchvariant ActRII polypeptides are provided throughout the presentdisclosure as well as in International Patent Application PublicationNos. WO 2006/012627, WO 2007/062188, WO 2008/097541, WO 2010/151426, andWO 2011/020045, which are incorporated herein by reference in theirentirety. Numbering of amino acids for all ActRII-related polypeptidesdescribed herein is based on the numbering of the human ActRII precursorprotein sequence provided below (SEQ ID NO: 1), unless specificallydesignated otherwise.

The canonical human ActRII precursor protein sequence is as follows:

(SEQ ID NO: 1)   1 MGAAAKLAFA VFLISCSSGA ILGRSETQEC LFFNANWEKD RT

QTGVEPC  51 YGDKDKRRHC FATWK

ISGS IEIVKQGCWL DDINCYDRTD CVEKKDSPEV 101YFCCCEGNMC NEKFSYFPEM EVTQPTSNPV TPKPPYYNIL LYSLVPLMLI 151AGIVICAFWV YRHHKMAYPP VLVPTQDPGP PPPSPLLGLK PLQLLEVKAR 201GRFGCVWKAQ LLNEYVAVKI FPIQDKQSWQ NEYEVYSLPG MKHENILQFI 251GAEKRGTSVD VDLWLITAFH EKGSLSDFLK ANVVSWNELC HIAETMARGL 301AYLHEDIPGL KDGHKPAISH RDIKSKNVLL KNNLTACIAD FGLALKFEAG 351KSAGDTHGQV GTRRYMAPEV LEGAINFQRD AFLRIDMYAM GLVLWELASR 401CTAADGPVDE YMLPFEEEIG QHPSLEDMQE VVVHKKKRPV LRDYWQKHAG 451MAMLCETIEE CWDHDAEARL SAGCVGERIT QMQRLTNIIT TEDIVTVVTM 501VTNVDFPPKE SSL

The signal peptide is indicated by a single underline; the extracellulardomain is indicated in bold font; and the potential, endogenous N-linkedglycosylation sites are indicated by a double underline.

A processed (mature) extracellular human ActRII polypeptide sequence isas follows:

(SEQ ID NO: 2) ILGRSETQECLEENANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFP EMEVTQPTSNPVTPKPP

The C-terminal “tail” of the extracellular domain is indicated by singleunderline. The sequence with the “tail” deleted (a Δ15 sequence) is asfollows:

(SEQ ID NO: 3) ILGRSETQECLEENANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFP EM

The nucleic acid sequence encoding human ActRII precursor protein isshown below (SEQ ID NO: 4), as follows nucleotides 159-1700 of GenbankReference Sequence NM_001616.4. The sequence is underlined.

(SEQ ID NO: 4) 1 ATGGGAGCTG CTGCAAAGTT GGCGTTTGCC GTCTTTCTTA TCTCCTGTTC51 TTCAGGTGCT ATACTTGGTA GATCAGAAAC TCAGGAGTGT CTTTTCTTTA 101ATGCTAATTG GGAAAAAGAC AGAACCAATC AAACTGGTGT TGAACCGTGT 151TATGGTGACA AAGATAAACG GCGGCATTGT TTTGCTACCT GGAAGAATAT 201TTCTGGTTCC ATTGAAATAG TGAAACAAGG TTGTTGGCTG GATGATATCA 251ACTGCTATGA CAGGACTGAT TGTGTAGAAA AAAAAGACAG CCCTGAAGTA 301TATTTTTGTT GCTGTGAGGG CAATATGTGT AATGAAAAGT TTTCTTATTT 351TCCGGAGATG GAAGTCACAC AGCCCACTTC AAATCCAGTT ACACCTAAGC 401CACCCTATTA CAACATCCTG CTCTATTCCT TGGTGCCACT TATGTTAATT 451GCGGGGATTG TCATTTGTGC ATTTTGGGTG TACAGGCATC ACAAGATGGC 501CTACCCTCCT GTACTTGTTC CAACTCAAGA CCCAGGACCA CCCCCACCTT 551CTCCATTACT AGGTTTGAAA CCACTGCAGT TATTAGAAGT GAAAGCAAGG 601GGAAGATTTG GTTGTGTCTG GAAAGCCCAG TTGCTTAACG AATATGTGGC 651TGTCAAAATA TTTCCAATAC AGGACAAACA GTCATGGCAA AATGAATACG 701AAGTCTACAG TTTGCCTGGA ATGAAGCATG AGAACATATT ACAGTTCATT 751GGTGCAGAAA AACGAGGCAC CAGTGTTGAT GTGGATCTTT GGCTGATCAC 801AGCATTTCAT GAAAAGGGTT CACTATCAGA CTTTCTTAAG GCTAATGTGG 851TCTCTTGGAA TGAACTGTGT CATATTGCAG AAACCATGGC TAGAGGATTG 901GCATATTTAC ATGAGGATAT ACCTGGCCTA AAAGATGGCC ACAAACCTGC 951CATATCTCAC AGGGACATCA AAAGTAAAAA TGTGCTGTTG AAAAACAACC 1001TGACAGCTTG CATTGCTGAC TTTGGGTTGG CCTTAAAATT TGAGGCTGGC 1051AAGTCTGCAG GCGATACCCA TGGACAGGTT GGTACCCGGA GGTACATGGC 1101TCCAGAGGTA TTAGAGGGTG CTATAAACTT CCAAAGGGAT GCATTTTTGA 1151GGATAGATAT GTATGCCATG GGATTAGTCC TATGGGAACT GGCTTCTCGC 1201TGTACTGCTG CAGATGGACC TGTAGATGAA TACATGTTGC CATTTGAGGA 1251GGAAATTGGC CAGCATCCAT CTCTTGAAGA CATGCAGGAA GTTGTTGTGC 1301ATAAAAAAAA GAGGCCTGTT TTAAGAGATT ATTGGCAGAA ACATGCTGGA 1351ATGGCAATGC TCTGTGAAAC CATTGAAGAA TGTTGGGATC ACGACGCAGA 1401AGCCAGGTTA TCAGCTGGAT GTGTAGGTGA AAGAATTACC CAGATGCAGA 1451GACTAACAAA TATTATTACC ACAGAGGACA TTGTAACAGT GGTCACAATG 1501GTGACAAATG TTGACTTTCC TCCCAAAGAA TCTAGTCTA 

The nucleic acid sequence encoding processed soluble (extracellular)human ActRII polypeptide is as follows:

(SEQ ID NO: 5) 1 ATACTTGGTA GATCAGAAAC TCAGGAGTGT CTTTTCTTTA ATGCTAATTG51 GGAAAAAGAC AGAACCAATC AAACTGGTGT TGAACCGTGT TATGGTGACA 101AAGATAAACG GCGGCATTGT TTTGCTACCT GGAAGAATAT TTCTGGTTCC 151ATTGAAATAG TGAAACAAGG TTGTTGGCTG GATGATATCA ACTGCTATGA 201CAGGACTGAT TGTGTAGAAA AAAAAGACAG CCCTGAAGTA TATTTTTGTT 251GCTGTGAGGG CAATATGTGT AATGAAAAGT TTTCTTATTT TCCGGAGATG 301GAAGTCACAC AGCCCACTTC AAATCCAGTT ACACCTAAGC CACCC 

ActRII is well-conserved among vertebrates, with large stretches of theextracellular domain completely conserved. For example, FIG. 2 depicts amulti-sequence alignment of a human ActRII extracellular domain comparedto various ActRII orthologs. Many of the ligands that bind to ActRII arealso highly conserved. Accordingly, from these alignments, it ispossible to predict key amino acid positions within the ligand-bindingdomain that are important for normal ActRII-ligand binding activities aswell as to predict amino acid positions that are likely to be tolerantto substitution without significantly altering normal ActRII-ligandbinding activities. Therefore, an active, human ActRII variantpolypeptide useful in accordance with the presently disclosed methodsmay include one or more amino acids at corresponding positions from thesequence of another vertebrate ActRII, or may include a residue that issimilar to that in the human or other vertebrate sequences.

An alignment of the amino acid sequences of human ActRIIA extracellulardomain and human ActRIIB extracellular domain are illustrated in FIG. 1. This alignment indicates amino acid residues within both receptorsthat are believed to directly contact ActRII ligands. For example, thecomposite ActRII structures indicated that the ActRIIA-ligand bindingpocket is defined, in part, by residues F31, N33, N35, K38 through T41,E47, Y50, K53 through K55, R57, H58, F60, T62, K74, W78 through N83,Y85, R87, E92, and K94 through F101. At these positions, it is expectedthat conservative mutations will be tolerated.

Without meaning to be limiting, the following examples illustrate thisapproach to defining an active ActRII variant. As illustrated in FIG. 2, F13 in the human extracellular domain is Y in Ovis aries (SEQ ID NO:7), Gallus gallus (SEQ ID NO: 10), Bos Taurus (SEQ ID NO: 36), Tyto alba(SEQ ID NO: 37), and Myotis davidii (SEQ ID NO: 38) ActRII, indicatingthat aromatic residues are tolerated at this position, including F, W,and Y. Q24 in the human extracellular domain is R in Bos Taurus ActRII,indicating that charged residues will be tolerated at this position,including D, R, K, H, and E. S95 in the human extracellular domain is Fin Gallus gallus and Tyto alba ActRII, indicating that this site may betolerant of a wide variety of changes, including polar residues, such asE, D, K, R, H, S, T, P, G, Y, and probably hydrophobic residue such asL, I, or F. E52 in the human extracellular domain is D in Ovis ariesActRII, indicating that acidic residues are tolerated at this position,including D and E. P29 in the human extracellular domain is relativelypoorly conserved, appearing as S in Ovis aries ActRII and L in Myotisdavidii ActRII, thus essentially any amino acid should be tolerated atthis position.

Moreover, as discussed above, ActRII proteins have been characterized inthe art in terms of structural/functional characteristics, particularlywith respect to ligand binding [Attisano et al. (1992) Cell68(1):97-108; Greenwald et al. (1999) Nature Structural Biology 6(1):18-22; Allendorph et al. (2006) PNAS 103 (20: 7643-7648; Thompson et al.(2003) The EMBO Journal 22(7): 1555-1566; as well as U.S. Pat. Nos.7,709,605, 7,612,041, and 7,842,663]. For example, a defining structuralmotif known as a three-finger toxin fold is important for ligand bindingby type I and type II receptors and is formed by conserved cysteineresidues located at varying positions within the extracellular domain ofeach monomeric receptor [Greenwald et al. (1999) Nat Struct Biol6:18-22; and Hinck (2012) FEBS Lett 586:1860-1870]. In addition to theteachings herein, these references provide amply guidance for how togenerate ActRII variants that retain one or more desired activities(e.g., ligand-binding activity).

For example, a defining structural motif known as a three-finger toxinfold is important for ligand binding by type I and type II receptors andis formed by conserved cysteine residues located at varying positionswithin the extracellular domain of each monomeric receptor [Greenwald etal. (1999) Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett586:1860-1870]. Accordingly, the core ligand-binding domains of humanActRII, as demarcated by the outermost of these conserved cysteines,corresponds to positions 30-110 of SEQ ID NO: 1 (ActRII precursor).Therefore, the structurally less-ordered amino acids flanking thesecysteine-demarcated core sequences can be truncated by about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, or 29 residues at the N-terminus and by about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 residues at the C-terminus without necessarily alteringligand binding. Exemplary ActRII extracellular domains truncationsinclude SEQ ID NOs: 2 and 3.

Accordingly, a general formula for an active portion (e.g., ligandbinding) of ActRII is a polypeptide that comprises, consists essentiallyof, or consists of amino acids 30-110 of SEQ ID NO: 1. Therefore ActRIIpolypeptides may, for example, comprise, consists essentially of, orconsists of an amino acid sequence that is at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to a portion of ActRII beginning at a residuecorresponding to any one of amino acids 21-30 (e.g., beginning at anyone of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) of SEQ IDNO: 1 and ending at a position corresponding to any one amino acids110-135 (e.g., ending at any one of amino acids 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, or 135) of SEQ ID NO: 1. Other examplesinclude constructs that begin at a position selected from 21-30 (e.g.,beginning at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30), 22-30 (e.g., beginning at any one of amino acids 22, 23, 24, 25,26, 27, 28, 29, or 30), 23-30 (e.g., beginning at any one of amino acids23, 24, 25, 26, 27, 28, 29, or 30), 24-30 (e.g., beginning at any one ofamino acids 24, 25, 26, 27, 28, 29, or 30) of SEQ ID NO: 1, and end at aposition selected from 111-135 (e.g., ending at any one of amino acids111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or 135), 112-135 (e.g.,ending at any one of amino acids 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134 or 135), 113-135 (e.g., ending at any one of amino acids 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134 or 135), 120-135 (e.g., ending at any oneof amino acids 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134 or 135), 130-135 (e.g., ending at any one of aminoacids 130, 131, 132, 133, 134 or 135), 111-134 (e.g., ending at any oneof amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or134), 111-133 (e.g., ending at any one of amino acids 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, or 133), 111-132 (e.g., ending at any oneof amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, or 132), or111-131 (e.g., ending at any one of amino acids 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, or 131) of SEQ ID NO: 1. Variants within these ranges are alsocontemplated, particularly those comprising, consisting essentially of,or consisting of an amino acid sequence that has at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identity to the corresponding portion of SEQ ID NO: 1.Thus, in some embodiments, an ActRII polypeptide may comprise, consistsessentially of, or consist of a polypeptide that is at least 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to amino acids 30-110 of SEQ ID NO: 1.Optionally, ActRII polypeptides comprise a polypeptide that is at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to amino acids 30-110 of SEQ IDNO: 1, and comprising no more than 1, 2, 5, 10 or 15 conservative aminoacid changes in the ligand-binding pocket.

In certain embodiments, the disclosure relates to an ActRII polypeptide,which includes fragments, functional variants, and modified formsthereof as well as uses thereof (e.g., treating, preventing, or reducingthe post-capillary pulmonary hypertension). Preferably, ActRIIpolypeptides are soluble (e.g., an extracellular domain of ActRII). Insome embodiments, ActRII polypeptides inhibit (e.g., Smad signaling) ofone or more GDF/BMP ligands [e.g., GDF11, GDF8, activin A, activin B,GDF3, BMP4, BMP6, BMP10, and/or BMP15]. In some embodiments, ActRIIpolypeptides bind to one or more GDF/BMP ligands [e.g., GDF11, GDF8,activin A, activin B, GDF3, BMP4, BMP6, BMP10, and/or BMP15]. In someembodiments, ActRII polypeptide of the disclosure comprise, consistessentially of, or consist of an amino acid sequence that is at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to a portion of ActRII beginningat a residue corresponding to amino acids 21-30 (e.g., beginning at anyone of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) of SEQ IDNO: 1 and ending at a position corresponding to any one amino acids110-135 (e.g., ending at any one of amino acids 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134 or 135) of SEQ ID NO: 1. In someembodiments, ActRII polypeptides comprise, consist, or consistessentially of an amino acid sequence that is at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical amino acids 30-110 of SEQ ID NO: 1. In certainembodiments, ActRII polypeptides comprise, consist, or consistessentially of an amino acid sequence that is at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical amino acids 21-135 of SEQ ID NO: 1. In someembodiments, ActRII polypeptides comprise, consist, or consistessentially of an amino acid sequence that is at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or100% identical to the amino acid sequence of any one of SEQ ID NOs: 1,2, 3, 23, 27, 30, and 41.

In some embodiments, ActRII polypeptides comprise, consist, or consistessentially of an amino acid sequence that is at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or100% identical to the amino acid sequence of SEQ ID NO: 23. In somealternative embodiments, the ActRII polypeptide (e.g., SEQ ID NO: 23)may lack the C-terminal lysine. In some embodiments, the ActRIIpolypeptide lacking the C-terminal lysine is SEQ ID NO: 41. In someembodiments, the ActRII polypeptides comprise, consist, or consistessentially of an amino acid sequence that is at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or100% identical to the amino acid sequence of SEQ ID NO: 41. In someembodiments, a patient is administered an ActRII polypeptide comprising,consisting, or consisting essentially of an amino acid sequence that isat least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence ofSEQ ID NO: 23. In some embodiments, a patient is administered an ActRIIpolypeptide comprising, consisting, or consisting essentially of anamino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical tothe amino acid sequence of SEQ ID NO: 41. In some embodiments, a patientis administered a combination of SEQ ID NO: 23 and SEQ ID NO: 41.

In certain aspects, the present disclosure relates to ActRIIpolypeptides. In some embodiments, ActRII traps of the presentdisclosure are variant ActRII polypeptides (e.g., ActRIIA polypeptides,ActRIIB polypeptides, or combinations thereof) that comprise one or moremutations (e.g., amino acid additions, deletions, substitutions, andcombinations thereof) in the extracellular domain (also referred to asthe ligand-binding domain) of an ActRII polypeptide (e.g., a “wild-type”or unmodified ActRII polypeptide) such that the variant ActRIIpolypeptide has one or more altered ligand-binding activities than thecorresponding wild-type ActRII polypeptide. In preferred embodiments,variant ActRII polypeptides of the present disclosure retain at leastone similar activity as a corresponding wild-type ActRII polypeptide.For example, preferable ActRII polypeptides bind to and inhibit (e.g.antagonize) the function of GDF11 and/or GDF8. In some embodiments,ActRII polypeptides of the present disclosure further bind to andinhibit one or more of ligand of the GDF/BMP [e.g., GDF11, GDF8, activinA, activin B, GDF3, BMP4, BMP6, BMP10, and/or BMP15]. Accordingly, thepresent disclosure provides ActRII polypeptides that have an alteredbinding specificity for one or more ActRII ligands.

To illustrate, one or more mutations may be selected that increase theselectivity of the altered ligand-binding domain for GDF11 and/or GDF8over one or more ActRII-binding ligands such as activins (activin A oractivin B), particularly activin A. Optionally, the alteredligand-binding domain has a ratio of K_(d) for activin binding to K_(d)for GDF11 and/or GDF8 binding that is at least 2-, 5-, 10-, 20-, 50-,100- or even 1000-fold greater relative to the ratio for the wild-typeligand-binding domain. Optionally, the altered ligand-binding domain hasa ratio of IC₅₀ for inhibiting activin to IC₅₀ for inhibiting GDF11and/or GDF8 that is at least 2-, 5-, 10-, 20-, 50-, 100- or even1000-fold greater relative to the wild-type ligand-binding domain.Optionally, the altered ligand-binding domain inhibits GDF11 and/or GDF8with an IC₅₀ at least 2-, 5-, 10-, 20-, 50-, 100- or even 1000-timesless than the IC₅₀ for inhibiting activin.

In certain embodiments, the present disclosure contemplates specificmutations of an ActRII polypeptide so as to alter the glycosylation ofthe polypeptide. Such mutations may be selected so as to introduce oreliminate one or more glycosylation sites, such as O-linked or N-linkedglycosylation sites. Asparagine-linked glycosylation recognition sitesgenerally comprise a tripeptide sequence, asparagine-X-threonine orasparagine-X-serine (where “X” is any amino acid) which is specificallyrecognized by appropriate cellular glycosylation enzymes. The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the polypeptide (forO-linked glycosylation sites). A variety of amino acid substitutions ordeletions at one or both of the first or third amino acid positions of aglycosylation recognition site (and/or amino acid deletion at the secondposition) results in non-glycosylation at the modified tripeptidesequence. Another means of increasing the number of carbohydratemoieties on a polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Depending on the coupling mode used, thesugar(s) may be attached to (a) arginine and histidine; (b) freecarboxyl groups; (c) free sulfhydryl groups such as those of cysteine;(d) free hydroxyl groups such as those of serine, threonine, orhydroxyproline; (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan; or (f) the amide group of glutamine. Removal ofone or more carbohydrate moieties present on a polypeptide may beaccomplished chemically and/or enzymatically.

Chemical deglycosylation may involve, for example, exposure of apolypeptide to the compound trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the amino acid sequence intact.Enzymatic cleavage of carbohydrate moieties on polypeptides can beachieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al. [Meth. Enzymol. (1987) 138:350]. Thesequence of a polypeptide may be adjusted, as appropriate, depending onthe type of expression system used, as mammalian, yeast, insect, andplant cells may all introduce differing glycosylation patterns that canbe affected by the amino acid sequence of the peptide. In general,polypeptides of the present disclosure for use in humans may beexpressed in a mammalian cell line that provides proper glycosylation,such as HEK293 or CHO cell lines, although other mammalian expressioncell lines are expected to be useful as well.

The present disclosure further contemplates a method of generatingmutants, particularly sets of combinatorial mutants of an ActRIIpolypeptide as well as truncation mutants. Pools of combinatorialmutants are especially useful for identifying functionally active (e.g.,GDF/BMP ligand binding) ActRII sequences. The purpose of screening suchcombinatorial libraries may be to generate, for example, polypeptidesvariants, which have altered properties, such as altered pharmacokineticor altered ligand binding. A variety of screening assays are providedbelow, and such assays may be used to evaluate variants. For example,ActRII variants may be screened for ability to bind to one or moreGDF/BMP ligands [e.g., GDF11, GDF8, activin A, activin B, GDF3, BMP4,BMP6, BMP10, and/or BMP15], to prevent binding of a GDF/BMP ligand to anActRII polypeptide, as well as heteromultimers thereof, and/or tointerfere with signaling caused by an GDF/BMP ligand.

The activity of ActRII polypeptides or variants thereof may also betested in a cell-based or in vivo assay. For example, the effect of anActRII polypeptide on the expression of genes involved in PcPHpathogenesis may be assessed. This may, as needed, be performed in thepresence of one or more recombinant ligand proteins [e.g., GDF11, GDF8,activin A, activin B, GDF3, BMP4, BMP6, BMP10, and/or BMP15], and cellsmay be transfected so as to produce an ActRII polypeptide, andoptionally, an GDF/BMP ligand. Likewise, an ActRII polypeptide may beadministered to a mouse or other animal and effects on PcPH pathogenesismay be assessed using art-recognized methods. Similarly, the activity ofan ActRII polypeptide or variant thereof may be tested in blood cellprecursor cells for any effect on growth of these cells, for example, bythe assays as described herein and those of common knowledge in the art.A SMAD-responsive reporter gene may be used in such cell lines tomonitor effects on downstream signaling.

Combinatorial-derived variants can be generated which have increasedselectivity or generally increased potency relative to a referenceActRII polypeptide. Such variants, when expressed from recombinant DNAconstructs, can be used in gene therapy protocols. Likewise, mutagenesiscan give rise to variants which have intracellular half-livesdramatically different than the corresponding unmodified ActRIIpolypeptide. For example, the altered protein can be rendered eithermore stable or less stable to proteolytic degradation or other cellularprocesses which result in destruction, or otherwise inactivation, of anunmodified polypeptide. Such variants, and the genes which encode them,can be utilized to alter polypeptide complex levels by modulating thehalf-life of the polypeptide. For instance, a short half-life can giverise to more transient biological effects and, when part of an inducibleexpression system, can allow tighter control of recombinant polypeptidecomplex levels within the cell. In an Fc fusion protein, mutations maybe made in the linker (if any) and/or the Fc portion to alter thehalf-life of the ActRII polypeptide.

A combinatorial library may be produced by way of a degenerate libraryof genes encoding a library of polypeptides which each include at leasta portion of potential ActRII polypeptide sequences. For instance, amixture of synthetic oligonucleotides can be enzymatically ligated intogene sequences such that the degenerate set of potential ActRII encodingnucleotide sequences are expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay).

There are many ways by which the library of potential homologs can begenerated from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be carried out in an automatic DNAsynthesizer, and the synthetic genes can then be ligated into anappropriate vector for expression. The synthesis of degenerateoligonucleotides is well known in the art [Narang, S A (1983)Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc. 3rdCleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevierpp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura etal. (1984) Science 198:1056; and Ike et al. (1983) Nucleic Acid Res.11:477]. Such techniques have been employed in the directed evolution ofother proteins [Scott et al., (1990) Science 249:386-390; Roberts et al.(1992) PNAS USA 89:2429-2433; Devlin et al. (1990) Science 249: 404-406;Cwirla et al., (1990) PNAS USA 87: 6378-6382; as well as U.S. Pat. Nos.5,223,409, 5,198,346, and 5,096,815].

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, ActRII polypeptides of thedisclosure can be generated and isolated from a library by screeningusing, for example, alanine scanning mutagenesis [Ruf et al. (1994)Biochemistry 33:1565-1572; Wang et al. (1994) J. Biol. Chem.269:3095-3099; Balint et al. (1993) Gene 137:109-118; Grodberg et al.(1993) Eur. J. Biochem. 218:597-601; Nagashima et al. (1993) J. Biol.Chem. 268:2888-2892; Lowman et al. (1991) Biochemistry 30:10832-10838;and Cunningham et al. (1989) Science 244:1081-1085], by linker scanningmutagenesis [Gustin et al. (1993) Virology 193:653-660; and Brown et al.(1992) Mol. Cell Biol. 12:2644-2652; McKnight et al. (1982) Science232:316], by saturation mutagenesis [Meyers et al., (1986) Science232:613]; by PCR mutagenesis [Leung et al. (1989) Method Cell Mol Biol1:11-19]; or by random mutagenesis, including chemical mutagenesis[Miller et al. (1992) A Short Course in Bacterial Genetics, CSHL Press,Cold Spring Harbor, N.Y.; and Greener et al. (1994) Strategies in MolBiol 7:32-34]. Linker scanning mutagenesis, particularly in acombinatorial setting, is an attractive method for identifying truncated(bioactive) forms of ActRII polypeptides.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of ActRII polypeptides. The most widely usedtechniques for screening large gene libraries typically comprise cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected. Preferred assays include ligand[e.g., GDF11, GDF8, activin A, activin B, GDF3, BMP4, BMP6, BMP10,and/or BMP15] binding assays and/or ligand-mediated cell signalingassays.

As will be recognized by one of skill in the art, most of the describedmutations, variants or modifications described herein may be made at thenucleic acid level or, in some cases, by post-translational modificationor chemical synthesis. Such techniques are well known in the art andsome of which are described herein. In part, the present disclosureidentifies functionally active portions (fragments) and variants ofActRII polypeptides that can be used as guidance for generating andusing other variant ActRII polypeptides within the scope of thedisclosure provided herein.

In certain embodiments, functionally active fragments of ActRIIpolypeptides of the present disclosure can be obtained by screeningpolypeptides recombinantly produced from the corresponding fragment ofthe nucleic acid encoding an ActRII polypeptide. In addition, fragmentscan be chemically synthesized using techniques known in the art such asconventional Merrifield solid phase f-Moc or t-Boc chemistry. Thefragments can be produced (recombinantly or by chemical synthesis) andtested to identify those peptidyl fragments that can function asantagonists (inhibitors) of ActRII receptors and/or one or more ligands[e.g., GDF11, GDF8, activin A, activin B, GDF3, BMP4, BMP6, BMP10,and/or BMP15].

In certain embodiments, ActRII polypeptides of the present disclosuremay further comprise post-translational modifications in addition to anythat are naturally present in the ActRII polypeptide. Such modificationsinclude, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation. As a result,the ActRII polypeptide may contain non-amino acid elements, such aspolyethylene glycols, lipids, polysaccharide or monosaccharide, andphosphates. Effects of such non-amino acid elements on the functionalityof a ligand trap polypeptide may be tested as described herein for otherActRII variants. When a polypeptide of the disclosure is produced incells by cleaving a nascent form of the polypeptide, post-translationalprocessing may also be important for correct folding and/or function ofthe protein. Different cells (e.g., CHO, HeLa, MDCK, 293, W138, NIH-3T3or HEK293) have specific cellular machinery and characteristicmechanisms for such post-translational activities and may be chosen toensure the correct modification and processing of the ActRIIpolypeptides.

In certain aspects, ActRII polypeptides of the present disclosureinclude fusion proteins having at least a portion (domain) of an ActRIIpolypeptide and one or more heterologous portions (domains). Well-knownexamples of such fusion domains include, but are not limited to,polyhistidine, Glu-Glu, glutathione S-transferase (GST), thioredoxin,protein A, protein G, an immunoglobulin heavy-chain constant region(Fc), maltose binding protein (MBP), or human serum albumin. A fusiondomain may be selected so as to confer a desired property. For example,some fusion domains are particularly useful for isolation of the fusionproteins by affinity chromatography. For the purpose of affinitypurification, relevant matrices for affinity chromatography, such asglutathione-, amylase-, and nickel- or cobalt-conjugated resins areused. Many of such matrices are available in “kit” form, such as thePharmacia GST purification system and the QIAexpress™ system (Qiagen)useful with (HIS₆) fusion partners. As another example, a fusion domainmay be selected so as to facilitate detection of the ActRII polypeptide.Examples of such detection domains include the various fluorescentproteins (e.g., GFP) as well as “epitope tags,” which are usually shortpeptide sequences for which a specific antibody is available. Well-knownepitope tags for which specific monoclonal antibodies are readilyavailable include FLAG, influenza virus haemagglutinin (HA), and c-myctags. In some cases, the fusion domains have a protease cleavage site,such as for Factor Xa or thrombin, which allows the relevant protease topartially digest the fusion proteins and thereby liberate therecombinant proteins therefrom. The liberated proteins can then beisolated from the fusion domain by subsequent chromatographicseparation. Other types of fusion domains that may be selected includemultimerizing (e.g., dimerizing, tetramerizing) domains and functionaldomains (that confer an additional biological function) including, forexample constant domains from immunoglobulins (e.g., Fc domains).

In certain aspects, ActRII polypeptides of the present disclosurecontain one or more modifications that are capable of “stabilizing” thepolypeptides. By “stabilizing” is meant anything that increases the invitro half-life, serum half-life, regardless of whether this is becauseof decreased destruction, decreased clearance by the kidney, or otherpharmacokinetic effect of the agent. For example, such modificationsenhance the shelf-life of the polypeptides, enhance circulatoryhalf-life of the polypeptides, and/or reduce proteolytic degradation ofthe polypeptides. Such stabilizing modifications include, but are notlimited to, fusion proteins (including, for example, fusion proteinscomprising an ActRII polypeptide domain and a stabilizer domain),modifications of a glycosylation site (including, for example, additionof a glycosylation site to a polypeptide of the disclosure), andmodifications of carbohydrate moiety (including, for example, removal ofcarbohydrate moieties from a polypeptide of the disclosure). As usedherein, the term “stabilizer domain” not only refers to a fusion domain(e.g., an immunoglobulin Fc domain) as in the case of fusion proteins,but also includes nonproteinaceous modifications such as a carbohydratemoiety, or nonproteinaceous moiety, such as polyethylene glycol. Incertain preferred embodiments, an ActRII polypeptide is fused with aheterologous domain that stabilizes the polypeptide (a “stabilizer”domain), preferably a heterologous domain that increases stability ofthe polypeptide in vivo. Fusions with a constant domain of animmunoglobulin (e.g., a Fc domain) are known to confer desirablepharmacokinetic properties on a wide range of proteins. Likewise,fusions to human serum albumin can confer desirable properties.

An example of a native amino acid sequence that may be used for the Fcportion of human IgG1 (G1Fc) is shown below (SEQ ID NO: 11). Dottedunderline indicates the hinge region, and solid underline indicatespositions with naturally occurring variants. In part, the disclosureprovides polypeptides comprising, consisting essential of, or consistingof amino acid sequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity toSEQ ID NO: 11. Naturally occurring variants in G1Fc would include E134Dand M136L according to the numbering system used in SEQ ID NO: 11 (seeUniprot P01857).

(SEQ ID NO: 11) 1

51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGK

Optionally, the IgG1 Fc domain has one or more mutations at residuessuch as Asp-265, lysine 322, and Asn-434. In certain cases, the mutantIgG1 Fc domain having one or more of these mutations (e.g., Asp-265mutation) has reduced ability of binding to the Fcγ receptor relative toa wild-type Fc domain. In other cases, the mutant Fc domain having oneor more of these mutations (e.g., Asn-434 mutation) has increasedability of binding to the MHC class I-related Fc-receptor (FcRN)relative to a wild-type IgG1 Fc domain.

An example of a native amino acid sequence that may be used for the Fcportion of human IgG2 (G2Fc) is shown below (SEQ ID NO: 12). Dottedunderline indicates the hinge region and double underline indicatespositions where there are data base conflicts in the sequence (accordingto UniProt P01859). In part, the disclosure provides polypeptidescomprising, consisting essential of, or consisting of amino acidsequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%,

(SEQ ID NO:12) 1

51 FNWYVDGVEV HNAKTKPREE QFNSTFRVVS VLTVVHQDWL NGKEYKCKVS 101NKGLPAPIEK TISKTKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP 151SDIAVEWESN GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVFS 201CSVMHEALHN HYTQKSLS LSPGK

Two examples of amino acid sequences that may be used for the Fe portionof human IgG3 (G3Fc) are shown below. The hinge region in G3Fc can be upto four times as long as in other Fc chains and contains three identical15-residue segments preceded by a similar 17-residue segment. The firstG3Fc sequence shown below (SEQ ID NO: 13) contains a short hinge regionconsisting of a single 15-residue segment, whereas the second G3Fcsequence (SEQ ID NO: 14) contains a full-length hinge region. In eachcase, dotted underline indicates the hinge region, and solid underlineindicates positions with naturally occurring variants according toUniProt P01859. In part, the disclosure provides polypeptidescomprising, consisting essential of, or consisting of amino acidsequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SE ID NOs: 13 and14.

(SEQ ID NO: 13) 1

51 VSHEDPEVQF KWYVDGVEVH NAKTKPREEQ YNSTFRVVSV LTVLHQDWLN 101GKEYKCKVSN KALPAPIEKT ISKTKGQPRE PQVYTLPPSR EEMTKNQVSL 151TCLVKGFYPS DIAVEWESSG QPENNYNTTP PMLDSDGSFF LYSKLTVDKS 201RWQQGNIFSC SVMHEALHNR FTQKSLSLSP GK (SEQ ID NO: 14) 1

51

101 EDPEVQFKWY VDGVEVHNAK TKPREEQYNS TFRVVSVLTV LHQDWLNGKE 151YKCKVSNKAL PAPIEKTISK TKGQPREPQV YTLPPSREEM TKNQVSLTCL 201VKGFYPSDIA VEWESSGQPE NNYNTTPPML DSDGSFFLYS KLTVDKSRWQ 251QGNIFSCSVM HEALHNRFTQ KSLSLSPGK

Naturally occurring variants in G3Fc (for example, see Uniprot P01860)include E68Q, P76L, E79Q, Y81F, D97N, N100D, T124A, S169N, S169del,F221Y when converted to the numbering system used in SEQ ID NO: 13, andthe present disclosure provides fusion proteins comprising G3Fc domainscontaining one or more of these variations. In addition, the humanimmunoglobulin IgG3 gene (IGHG3) shows a structural polymorphismcharacterized by different hinge lengths [see Uniprot P01859].Specifically, variant WIS is lacking most of the V region and all of theCHi region. It has an extra interchain disulfide bond at position 7 inaddition to the 11 normally present in the hinge region. Variant ZUClacks most of the V region, all of the CHi region, and part of thehinge. Variant OMM may represent an allelic form or another gamma chainsubclass. The present disclosure provides additional fusion proteinscomprising G3Fc domains containing one or more of these variants.

An example of a native amino acid sequence that may be used for the Fcportion of human IgG4 (G4Fc) is shown below (SEQ ID NO: 15). Dottedunderline indicates the hinge region. In part, the disclosure providespolypeptides comprising, consisting essential of, or consisting of aminoacid sequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:15.

(SEQ ID NO: 15) 1

51 EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE 101YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL 151VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ 201EGNVFSCSVM HEALHNHYTQ KSLSLSLGK

A variety of engineered mutations in the Fc domain are presented hereinwith respect to the G1Fc sequence (SEQ ID NO: 11), and analogousmutations in G2Fc, G3Fc, and G4Fc can be derived from their alignmentwith G1Fc in FIG. 4 . Due to unequal hinge lengths, analogous Fcpositions based on isotype alignment (FIG. 4 ) possess different aminoacid numbers in SEQ ID NOs: 11, 12, 13, 14, and 15. It can also beappreciated that a given amino acid position in an immunoglobulinsequence consisting of hinge, C_(H)2, and C_(H)3 regions (e.g., SEQ IDNOs: 11, 12, 13, 14, and 15) will be identified by a different numberthan the same position when numbering encompasses the entire IgG1heavy-chain constant domain (consisting of the C_(H)1, hinge, C_(H)2,and C_(H)3 regions) as in the Uniprot database. For example,correspondence between selected C_(H)3 positions in a human G1Fcsequence (SEQ ID NO: 11), the human IgG1 heavy chain constant domain(Uniprot P01857), and the human IgG1 heavy chain is as follows.

Correspondence of C_(H)3 Positions in Different Numbering Systems G1FcIgG1 heavy chain IgG1 heavy chain (Numbering begins constant domain (EUnumbering at first threonine in (Numbering begins at scheme of Kabathinge region) C_(H)1) et al., 1991*) Y127 Y232 Y349 S132 S237 S354 E134E239 E356 T144 T249 T366 L146 L251 L368 K170 K275 K392 D177 D282 D399Y185 Y290 Y407 K187 K292 K409 *Kabat et al. (eds) 1991; pp. 688-696 inSequences of Proteins of Immunological Interest, 5^(th) ed., Vol. 1,NIH, Bethesda, MD.

Various methods are known in the art that increase desired pairing ofFc-containing fusion polypeptide chains in a single cell line to producea preferred asymmetric fusion protein at acceptable yields [Klein et al(2012) mAbs 4:653-663; and Spiess et al (2015) Molecular Immunology67(2A): 95-106]. Methods to obtain desired pairing of Fc-containingchains include, but are not limited to, charge-based pairing(electrostatic steering), “knobs-into-holes” steric pairing, SEEDbodypairing, and leucine zipper-based pairing [Ridgway et al (1996) ProteinEng 9:617-621; Merchant et al (1998) Nat Biotech 16:677-681; Davis et al(2010) Protein Eng Des Sel 23:195-202; Gunasekaran et al (2010);285:19637-19646; Wranik et al (2012) J Biol Chem 287:43331-43339; U.S.Pat. No. 5,932,448; WO 1993/011162; WO 2009/089004, and WO 2011/034605].

It is understood that different elements of the fusion proteins (e.g.,immunoglobulin Fc fusion proteins) may be arranged in any manner that isconsistent with desired functionality. For example, an ActRIIpolypeptide domain may be placed C-terminal to a heterologous domain, oralternatively, a heterologous domain may be placed C-terminal to anActRII polypeptide domain. The ActRII polypeptide domain and theheterologous domain need not be adjacent in a fusion protein, andadditional domains or amino acid sequences may be included C- orN-terminal to either domain or between the domains.

For example, an ActRII receptor fusion protein may comprise an aminoacid sequence as set forth in the formula A-B-C. The B portioncorresponds to an ActRII polypeptide domain. The A and C portions may beindependently zero, one, or more than one amino acid, and both the A andC portions when present are heterologous to B. The A and/or C portionsmay be attached to the B portion via a linker sequence. A linker may berich in glycine (e.g., 2-10, 2-5, 2-4, 2-3 glycine residues) or glycineand proline residues and may, for example, contain a single sequence ofthreonine/serine and glycines or repeating sequences of threonine/serineand/or glycines, e.g., GGG (SEQ ID NO: 16), GGGG (SEQ ID NO: 17), TGGGG(SEQ ID NO: 18), SGGGG (SEQ ID NO: 19), TGGG (SEQ ID NO: 20), SGGG (SEQID NO: 21), or GGGGS (SEQ ID NO: 22) singlets, or repeats. In certainembodiments, an ActRII fusion protein comprises an amino acid sequenceas set forth in the formula A-B-C, wherein A is a leader (signal)sequence, B consists of an ActRII polypeptide domain, and C is apolypeptide portion that enhances one or more of in vivo stability, invivo half-life, uptake/administration, tissue localization ordistribution, formation of protein complexes, and/or purification. Incertain embodiments, an ActRII fusion protein comprises an amino acidsequence as set forth in the formula A-B-C, wherein A is a TPA leadersequence, B consists of an ActRII receptor polypeptide domain, and C isan immunoglobulin Fc domain. Preferred fusion proteins comprise theamino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 30, and41.

In preferred embodiments, ActRII polypeptides to be used in accordancewith the methods described herein are isolated polypeptides. As usedherein, an isolated protein or polypeptide is one which has beenseparated from a component of its natural environment. In someembodiments, a polypeptide of the disclosure is purified to greater than95%, 96%, 97%, 98%, or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). Methods for assessment of purity are well known in the art [see,e.g., Flatman et al., (2007) J. Chromatogr. B 848:79-87]. In someembodiments, ActRII polypeptides to be used in accordance with themethods described herein are recombinant polypeptides.

ActRII polypeptides of the disclosure can be produced by a variety ofart-known techniques. For example, polypeptides of the disclosure can besynthesized using standard protein chemistry techniques such as thosedescribed in Bodansky, M. Principles of Peptide Synthesis, SpringerVerlag, Berlin (1993) and Grant G. A. (ed.), Synthetic Peptides: AUser's Guide, W. H. Freeman and Company, New York (1992). In addition,automated peptide synthesizers are commercially available (e.g.,Advanced ChemTech Model 396; Milligen/Biosearch 9600). Alternatively,the polypeptides of the disclosure, including fragments or variantsthereof, may be recombinantly produced using various expression systems[e.g., E. coli, Chinese Hamster Ovary (CHO) cells, COS cells,baculovirus] as is well known in the art. In a further embodiment, themodified or unmodified polypeptides of the disclosure may be produced bydigestion of recombinantly produced full-length ActRII polypeptides byusing, for example, a protease, e.g., trypsin, thermolysin,chymotrypsin, pepsin, or paired basic amino acid converting enzyme(PACE). Computer analysis (using commercially available software, e.g.,MacVector, Omega, PCGene, Molecular Simulation, Inc.) can be used toidentify proteolytic cleavage sites. Alternatively, such polypeptidesmay be produced from recombinantly generated full-length ActRIIpolypeptides using chemical cleavage (e.g., cyanogen bromide,hydroxylamine, etc.).

3. Nucleic Acids Encoding ActRII Polypeptides

In certain embodiments, the present disclosure provides isolated and/orrecombinant nucleic acids encoding ActRII polypeptides (includingfragments, functional variants, and fusion proteins thereof).

As used herein, isolated nucleic acid(s) refers to a nucleic acidmolecule that has been separated from a component of its naturalenvironment. An isolated nucleic acid includes a nucleic acid moleculecontained in cells that ordinarily contain the nucleic acid molecule,but the nucleic acid molecule is present extrachromosomally or at achromosomal location that is different from its natural chromosomallocation.

In certain embodiments, nucleic acids encoding ActRII polypeptides ofthe disclosure are understood to include nucleic acids that are variantsof any one of SEQ ID NOs: 4, 5, or 28. Variant nucleotide sequencesinclude sequences that differ by one or more nucleotide substitutions,additions, or deletions including allelic variants, and therefore, willinclude coding sequence that differ from the nucleotide sequencedesignated in any one of SEQ ID NOs: 4, 5, or 28.

In certain embodiments, ActRII polypeptides of the disclosure areencoded by isolated and/or recombinant nucleic acid sequences that areat least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%,99%, or 100% identical to any one of SEQ ID NOs: 4, 5, or 28. One ofordinary skill in the art will appreciate that nucleic acid sequencesthat are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%,97%, 98%, 99%, or 100% identical to the sequences complementary to SEQID NOs: 4, 5, or 28, and variants thereof, are also within the scope ofthe present disclosure. In further embodiments, the nucleic acidsequences of the disclosure can be isolated, recombinant, and/or fusedwith a heterologous nucleotide sequence, or in a DNA library.

In other embodiments, nucleic acids of the present disclosure alsoinclude nucleotide sequences that hybridize under highly stringentconditions to the nucleotide sequence designated in SEQ ID NOs: 4, 5, or28, complement sequences of SEQ ID NOs: 4, 5, or 28, or fragmentsthereof. As discussed above, one of ordinary skill in the art willunderstand readily that appropriate stringency conditions which promoteDNA hybridization can be varied. One of ordinary skill in the art willunderstand readily that appropriate stringency conditions which promoteDNA hybridization can be varied. For example, one could perform thehybridization at 6.0× sodium chloride/sodium citrate (SSC) at about 45°C., followed by awash of 2.0×SSC at 50° C. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In one embodiment, the disclosureprovides nucleic acids which hybridize under low stringency conditionsof 6×SSC at room temperature followed by awash at 2×SSC at roomtemperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NOs: 4, 5, or 28 to degeneracy in the genetic code are alsowithin the scope of the disclosure. For example, a number of amino acidsare designated by more than one triplet. Codons that specify the sameamino acid, or synonyms (for example, CAU and CAC are synonyms forhistidine) may result in “silent” mutations which do not affect theamino acid sequence of the protein. However, it is expected that DNAsequence polymorphisms that do lead to changes in the amino acidsequences of the subject proteins will exist among mammalian cells. Oneskilled in the art will appreciate that these variations in one or morenucleotides (up to about 3-5% of the nucleotides) of the nucleic acidsencoding a particular protein may exist among individuals of a givenspecies due to natural allelic variation. Any and all such nucleotidevariations and resulting amino acid polymorphisms are within the scopeof this disclosure.

In certain embodiments, the recombinant nucleic acids of the presentdisclosure may be operably linked to one or more regulatory nucleotidesequences in an expression construct. Regulatory nucleotide sequenceswill generally be appropriate to the host cell used for expression.Numerous types of appropriate expression vectors and suitable regulatorysequences are known in the art and can be used in a variety of hostcells. Typically, one or more regulatory nucleotide sequences mayinclude, but are not limited to, promoter sequences, leader or signalsequences, ribosomal binding sites, transcriptional start andtermination sequences, translational start and termination sequences,and enhancer or activator sequences. Constitutive or inducible promotersas known in the art are contemplated by the disclosure. The promotersmay be either naturally occurring promoters, or hybrid promoters thatcombine elements of more than one promoter. An expression construct maybe present in a cell on an episome, such as a plasmid, or the expressionconstruct may be inserted in a chromosome. In some embodiments, theexpression vector contains a selectable marker gene to allow theselection of transformed host cells. Selectable marker genes are wellknown in the art and can vary with the host cell used.

In certain aspects, the subject nucleic acid disclosed herein isprovided in an expression vector comprising a nucleotide sequenceencoding an ActRII polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the ActRII polypeptide. Accordingly,the term regulatory sequence includes promoters, enhancers, and otherexpression control elements. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press, San Diego, Calif. (1990). For instance, any of a widevariety of expression control sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express DNA sequences encoding an ActRII polypeptide. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, RSV promoters, the lac system, the trp system, the TACor TRC system, T7 promoter whose expression is directed by T7 RNApolymerase, the major operator and promoter regions of phage lambda, thecontrol regions for fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

A recombinant nucleic acid of the present disclosure can be produced byligating the cloned gene, or a portion thereof, into a vector suitablefor expression in either prokaryotic cells, eukaryotic cells (yeast,avian, insect or mammalian), or both. Expression vehicles for productionof a recombinant ActRII polypeptide include plasmids and other vectors.For instance, suitable vectors include plasmids of the following types:pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids,pBTac-derived plasmids and pUC-derived plasmids for expression inprokaryotic cells, such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, e.g., Molecular Cloning A Laboratory Manual, 3rdEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 2001). In some instances, it may be desirable toexpress the recombinant polypeptides by the use of a baculovirusexpression system. Examples of such baculovirus expression systemsinclude pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors(such as the β-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject ActRII polypeptides in CHO cells, such as a Pcmv-Scriptvector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen,Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wis.). As willbe apparent, the subject gene constructs can be used to cause expressionof the subject ActRII polypeptides in cells propagated in culture, e.g.,to produce proteins, including fusion proteins or variant proteins, forpurification.

This disclosure also pertains to a host cell transfected with arecombinant gene including a coding sequence for one or more of thesubject ActRII polypeptides. The host cell may be any prokaryotic oreukaryotic cell. For example, an ActRII polypeptide of the disclosuremay be expressed in bacterial cells such as E. coli, insect cells (e.g.,using a baculovirus expression system), yeast, or mammalian cells [e.g.a Chinese hamster ovary (CHO) cell line]. Other suitable host cells areknown to those skilled in the art.

Accordingly, the present disclosure further pertains to methods ofproducing the subject ActRII polypeptides. For example, a host celltransfected with an expression vector encoding an ActRII polypeptide canbe cultured under appropriate conditions to allow expression of theActRII polypeptide to occur. The polypeptide may be secreted andisolated from a mixture of cells and medium containing the polypeptide.Alternatively, the ActRII polypeptide may be retained cytoplasmically orin a membrane fraction and the cells harvested, lysed and the proteinisolated. A cell culture includes host cells, media and otherbyproducts. Suitable media for cell culture are well known in the art.The subject polypeptides can be isolated from cell culture medium, hostcells, or both, using techniques known in the art for purifyingproteins, including ion-exchange chromatography, gel filtrationchromatography, ultrafiltration, electrophoresis, immunoaffinitypurification with antibodies specific for particular epitopes of theActRII polypeptides, and affinity purification with an agent that bindsto a domain fused to the ActRII polypeptide (e.g., a protein A columnmay be used to purify an ActRII-Fc fusion proteins). In someembodiments, the ActRII polypeptide is a fusion protein containing adomain which facilitates its purification.

In some embodiments, purification is achieved by a series of columnchromatography steps, including, for example, three or more of thefollowing, in any order: protein A chromatography, Q sepharosechromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange. An ActRIIprotein may be purified to a purity of >90%, >95%, >96%, >98%, or >99%as determined by size exclusion chromatographyand >90%, >95%, >96%, >98%, or >99% as determined by SDS PAGE. Thetarget level of purity should be one that is sufficient to achievedesirable results in mammalian systems, particularly non-human primates,rodents (mice), and humans.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant ActRIIpolypeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified ActRII polypeptide. See, e.g.,Hochuli et al. (1987) J. Chromatography 411:177; and Janknecht et al.(1991) PNAS USA 88:8972.

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence. See,e.g., Current Protocols in Molecular Biology, eds. Ausubel et al., JohnWiley & Sons: 1992.

4. Methods of Use

In part, the present disclosure relates to methods of treatingpost-capillary pulmonary hypertension (PcPH) (e.g., WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide as described herein. In someembodiments, the PcPH is combined post- and pre-capillary PH. In certainembodiments, the present disclosure provides methods of treating orpreventing post-capillary pulmonary hypertension (PcPH) in an individualin need thereof through administering to the individual atherapeutically effective amount of an ActRII polypeptide as describedherein. These methods are particularly aimed at therapeutic andprophylactic treatments of animals, and more particularly, humans. Theterms “subject,” an “individual,” or a “patient” are interchangeablethroughout the specification and refer to either a human or a non-humananimal. These terms include mammals, such as humans, non-human primates,laboratory animals, livestock animals (including bovines, porcines,camels, etc.), companion animals (e.g., canines, felines, otherdomesticated animals, etc.) and rodents (e.g., mice and rats). Inparticular embodiments, the patient, subject or individual is a human.

The terms “treatment”, “treating”, “alleviating” and the like are usedherein to generally mean obtaining a desired pharmacologic and/orphysiologic effect, and may also be used to refer to improving,alleviating, and/or decreasing the severity of one or more clinicalcomplication of a condition being treated (e.g., WHO Group 2 and/orGroup 5 PH). The effect may be prophylactic in terms of completely orpartially delaying the onset or recurrence of a disease, condition, orcomplications thereof, and/or may be therapeutic in terms of a partialor complete cure for a disease or condition and/or adverse effectattributable to the disease or condition. “Treatment” as used hereincovers any treatment of a disease or condition of a mammal, particularlya human. As used herein, a therapeutic that “prevents” a disorder orcondition refers to a compound that, in a statistical sample, reducesthe occurrence of the disorder or condition in a treated sample relativeto an untreated control sample, or delays the onset of the disease orcondition, relative to an untreated control sample.

In general, treatment or prevention of a disease or condition asdescribed in the present disclosure (e.g., WHO Group 2 and/or Group 5PH) is achieved by administering one or more ActRII polypeptides of thepresent disclosure in an “effective amount”. An effective amount of anagent refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result. A“therapeutically effective amount” of an agent of the present disclosuremay vary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the agent to elicit adesired response in the individual. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result.

In certain aspects, the disclosure contemplates the use of an ActRIIpolypeptide, in combination with one or more additional active agents orother supportive therapy for treating or preventing a disease orcondition (e.g., WHO Group 2 and/or Group 5 PH). As used herein, “incombination with”, “combinations of”, “combined with”, or “conjoint”administration refers to any form of administration such that additionalactive agents or supportive therapies (e.g., second, third, fourth,etc.) are still effective in the body (e.g., multiple compounds aresimultaneously effective in the patient for some period of time, whichmay include synergistic effects of those compounds). Effectiveness maynot correlate to measurable concentration of the agent in blood, serum,or plasma. For example, the different therapeutic compounds can beadministered either in the same formulation or in separate formulations,either concomitantly or sequentially, and on different schedules. Thus,a subject who receives such treatment can benefit from a combined effectof different active agents or therapies. One or more ActRII polypeptidesof the disclosure can be administered concurrently with, prior to, orsubsequent to, one or more other additional agents or supportivetherapies, such as those disclosed herein. In general, each active agentor therapy will be administered at a dose and/or on a time scheduledetermined for that particular agent. The particular combination toemploy in a regimen will take into account compatibility of the ActRIIpolypeptide of the present disclosure with the additional active agentor therapy and/or the desired effect.

WHO Classification Outline

A pulmonary hypertension condition treated by methods describe herein,can comprise any one or more of the conditions recognized according tothe World Health Organization (WHO). See, e.g., Simonneau (2019) EurRespir J: 53:1801913.

TABLE 1 Clinical Classification of Pulmonary Hypertension Group 1:Pulmonary arterial hypertension (PAH)  1.1 Idiopathic PAH  1.2 HeritablePAH   1.2.1 BMPR2   1.2 2 ALK-1, ENG, SMAD9, CAV1, KCNK3   1.2.3 Unknown 1.3 Drug and toxin induced PAH  1.4 Associated with:   1.4.1 Connectivetissue disease   1.4.2 HIV infection   1.4.3 Portal hypertension   1.4.4Congenital heart diseases   1.4.5 Schistosomiasis  1.5 PAH long-termresponders to calcium channel blockers  1.6 PAH with overt features ofvenous/capillaries (PVOD/PCH) involvement  1.7 Persistent PH of thenewborn syndrome Group 2: Pulmonary hypertension due to left heartdisease  2.1 PH due to heart failure with preserved LVEF¹ (HFpEF)  2.2PH due to heart failure with reduced LVEF (HFrEF)  2.3 Valvular heartdisease  2.4 Congenital/acquired cardiovascular conditions leading topost-capillary PH Group 3: Pulmonary hypertension due to lung diseaseand/or hypoxia  3.1 Obstructive lung disease  3.2 Restrictive lungdisease  3.3 Other lung disease with mixed restrictive/obstructivepattern  3.4 Hypoxia without lung disease  3.5 Developmental lungdisorders Group 4: Pulmonary hypertension due to pulmonary arteryobstructions  4.1 Chronic thromboembolic PH  4.2 Other pulmonary arteryobstructions   4.2.1 Sarcoma (high or intermediate grade) orangiosarcoma   4.2.2 Other malignant tumours    Renal carcinoma   Uterine carcinoma    Germ cell tumours of the testis    Other tumours  4.2.3 Non-malignant tumours    Uterine leiomyoma   4.2.4 Arteritiswithout connective tissue disease   4.2.5 Congenital pulmonary arterystenoses   4.2.6 Parasites    Hydatidosis Group 5: Pulmonaryhypertension with unclear and/or multifactorial mechanisms.  5.1Hematological disorders (e.g., Chronic hemolytic anaemia andmyeloproliferative  disorders)  5.2 Systemic and metabolic disorders(e.g., Pulmonary Langerhans cell histiocytosis,  Gaucher disease,Glycogen storage disease, Neurofibromatosis, and Sarcoidosis)  5.3Others (e.g., Chronic renal failure with or without haemodialysis andFibrosing  mediastinitis)  5.4 Complex congenital heart disease ¹Leftventricular ejection fraction

The clinical purpose of the classification of PH is to categorizeclinical conditions associated with PH into five groups according totheir pathophysiological mechanisms, clinical presentation, hemodynamiccharacteristics, and treatment strategy. This clinical classificationmay be updated when new data are available on the above features or whenadditional clinical entities are considered.

Pulmonary hypertension (PH) has been previously classified as primary orsecondary PH. The term primary pulmonary hypertension has now beenreplaced by idiopathic PAH or familial PAH depending on the absence orpresence of genetic information; the term secondary pulmonaryhypertension has been abandoned.

As used herein, the term “pulmonary hemodynamic parameter” refers to anyparameter used to describe or evaluate the blood flow through the heartand pulmonary vasculature. Examples of pulmonary hemodynamic parametersinclude, but are not limited to, mean pulmonary artery pressure (mPAP),diastolic pulmonary artery pressure (dPAP) [also known as pulmonaryartery diastolic pressure (PADP)], systolic pulmonary artery pressure(sPAP) [also known as pulmonary artery systolic pressure (PASP)], meanright atrial pressure (mRAP), pulmonary capillary wedge pressure (PCWP)[also known as pulmonary artery wedge pressure (PAWP)], left ventricularend-diastolic pressure (LVEDP), diastolic pressure gradient (DPG) [alsoknown as diastolic pressure difference (DPD)], left atrial pressure(LAP), transpulmonary gradient (TPG), pulmonary vascular resistance(PVR) and cardiac output (CO).

Many of the pulmonary hemodynamic parameters described above areinterrelated. For example, PCWP is often used as a more convenient, lessinvasive approximation of LAP.

As another example, PVR is related to mPAP, PCWP and CO according to thefollowing equation:

PVR=(mPAP−PCWP)/CO[Woods Units]

The PVR measures the resistance to flow imposed by the pulmonaryvasculature without the influence of the left-sided filling pressure.PVR can also be measured according to the following equations:

PVR=TPG×80/CO[unit:dynes-sec-cm⁻⁵] ORPVR=(mPAP−PCWP)×80/CO[unit:dynes-sec-cm⁻⁵]

In some embodiments, the total PVR can be measured using the followingequation:

TPR=mPAP/CO

According to some embodiments, a pre-capillary pulmonary arterialcontribution to PH may be reflected by an elevated PVR. In someembodiments, the normal PVR is 20-130 dynes-sec-cm⁻⁵ or 0.5-1.1 Woodunits. According to some embodiments, an elevated PVR may refer to a PVRabove 2 Wood units, above 2.5 Wood units, above 3 Wood units or above3.5 Wood units.

As yet another example, TPG is the difference between mPAP and leftatrial pressure (PLA; commonly estimated by pulmonary capillary wedgepressure: PCWP) as shown by the following equation: TPG=mPAP-PCWP

The TPG is influenced by all the determinants of mPAP, including flow,resistance and left heart filling pressure. A pre-capillary pulmonaryarterial contribution to PH may be reflected by an increasedtrans-pulmonary gradient (TPG). According to some embodiments, anincreased TPG may refer to an mPAP-PCWP that exceeds 12-15 mmHg.

DPG (defined as diastolic PAP—mean PAWP) appears to best approach thecharacteristics required to determine pulmonary vascular disease. Insome embodiments, the DPG is synonymous with diastolic pressuredifference (DPD). In normal subjects, DPG generally lies in the 1-3 mmHgrange, and in patients evaluated for cardiac disease (excluding shunts),DPG remains ≤5 mmHg in most cases.

As a further example, mPAP is related to dPAP and sPAP according to thefollowing equation: mPAP=(⅔)dPAP+(⅓)sPAP

Furthermore, dPAP and sPAP can be used to calculate the pulse pressure(mmHg) using the following equation: pulse pressure=sPAP−dPAP

Pulse pressure can be used to calculate the pulmonary artery complianceusing the following equation: pulmonary artery compliance(mI.mmHg⁻¹)=stroke volume/pulse pressure. In some embodiments, thepulmonary hemodynamic parameters are measured directly, such as during aright heart catheterization. In other embodiments, the pulmonaryhemodynamic parameters are estimated and/or evaluated through othertechniques such as magnetic resonance imaging (MRI) or echocardiography.

Exemplary pulmonary hemodynamic parameters include mPAP, PAWP, TPG, DPG,and PVR. The one or more pulmonary hemodynamic parameters may bemeasured by any appropriate procedures, such as by utilizing a rightheart catheterization or echocardiography. Various hemodynamic types ofPH are shown in Table 2 together with their corresponding clinicalclassification (Table 1).

TABLE 2 Hemodynamic Types of Pulmonary Hypertension (PH) Hemo- Hemo-dynamic dynamic WHO PH Type Subtype Characteristics ClassificationPulmonary — mPAP > 20 mmHg All (Groups 1-5) Hypertension Pre-Capillary —mPAP > 20 mmHg Group 1: Pulmonary PH PAWP ≤ 15 mmHg lung disease and/orPVR ≥ 3 Wood units hypoxia Group 4: PH due to pulmonary arteryobstructions Group 5: PH with unclear and/or multifactorial mechanisms.arterial hypertension Group 3: PH due to Post- Isolated Post- mPAP > 20mmHg Group 2: PH due to Capillary Capillary PH PAWP > 15 mmHg left heartdisease PH PVR < 3 Wood units Group 5: PH with DPG < 7 mmHg unclearand/or Combined Pre- mPAP > 20 mmHg multifactorial and Post- PAWP > 15mmHg mechanisms. Capillary PH PVR ≥ 3 Wood units DPG ≥ 7 mmHg

The types of PH and the difference between pre-capillary pulmonaryhypertension and post-capillary pulmonary hypertension are based onpulmonary hemodynamic parameters. As used herein, the term“pre-capillary pulmonary hypertension” includes WHO clinical Groups 1,3, 4, and 5. In general, pre-capillary pulmonary hypertension ischaracterized using the pulmonary hemodynamic parameters shown in Table2 (i.e., an mPAP >20 mmHg or in some embodiments an mPAP >25 mmHg). Asused herein, the term “post-capillary pulmonary hypertension” (PcPH)includes both isolated post-capillary pulmonary hypertension (IpcPH) andcombined pre- and post-capillary pulmonary hypertension (CpcPH), bothwithin WHO clinical Groups 2 and 5. In some embodiments, IpcPH ischaracterized using the pulmonary hemodynamic parameters shown in Table2 (i.e., one or more of the following pulmonary hemodynamic parameters:mPAP >20 mmHg, PAWP >15 mmHg, PVR<3 Wood units, and/or DPG <7 mmHg). Insome embodiments, CpcPH is characterized using the pulmonary hemodynamicparameters shown in Table 2 (i.e., one or more of the followingpulmonary hemodynamic parameters: mPAP>20 mmHg, PAWP>15 mmHg, PVR≥3 Woodunits, and/or DPG≥7 mmHg). In some embodiments, CpcPH is characterizedas comprising one or more of the following hemodynamic parameters:mPAP≥25 mmHg; PAWP>15 mmHg; and PVR>3 WU.

The clinical classification or hemodynamic types of PH described hereinand the associated diagnostic parameters may be updated or varied basedon the availability of new or existing sources of data or whenadditional clinical entities are considered.

Characteristics of PH

The diagnosis of PH, including WHO PH class and functional group, can bedetermined based on symptoms and physical examination using a review ofa comprehensive set of parameters to determine if the hemodynamic andother criteria are met. Some of the criteria which may consideredinclude the patient's clinical presentation (e.g., shortness of breath,fatigue, weakness, angina, syncope, dry-couch, exercise-induced nauseaand vomiting), electrocardiogram (ECG) results, chest radiographresults, pulmonary function tests, arterial blood gases,echocardiography results, ventilation/perfusion lung scan results,high-resolution computed tomography results, contrast-enhanced computedtomography results, pulmonary angiography results, cardiac magneticresonance imaging, blood tests (e.g., biomarkers such as BNP orNT-proBNP), immunology, abdominal ultrasound scan, right heartcatherization (RHC), vasoreactivity, and genetic testing. See, e.g.,Galie N., et al Euro Heart J. (2016) 37, 67-119.

In some embodiments, a biomarker may be used to determine the diagnosisof PH. For instance, in some embodiments, the biomarker is a marker ofvascular dysfunction (e.g., asymmetric dimethylarginine (ADMA),endothelin-1, angiopoeitins, or von Willebrand factor). In someembodiments, the biomarker is a marker of inflammation (C-reactiveprotein, interleukin 6, chemokines). In some embodiments, the biomarkeris a marker of myocardial stress (e.g., (atrial natriuretic peptide,brain natriuretic peptide (BNP)/NT-proBNP, or troponins). In someembodiments, the biomarker is a marker of low CO and/or tissue hypoxia(e.g., pCO2, uric acid, growth differentiation factor 15 (GDF15), orosteopontin). In some embodiments, the biomarker is a marker ofsecondary organ damage (e.g., creatinine or bilirubin). See, e.g., GalieN., et al Euro Heart J. (2016) 37, 67-119.

Group 1 PH

Pulmonary arterial hypertension (WHO Group 1 PH) is a serious,progressive and life-threatening disease of the pulmonary vasculature,characterized by profound vasoconstriction and an abnormal proliferationof smooth muscle cells in the walls of the pulmonary arteries. Severeconstriction of the blood vessels in the lungs leads to very highpulmonary arterial pressures. These high pressures make it difficult forthe heart to pump blood through the lungs to be oxygenated. Patientswith PAH suffer from extreme shortness of breath as the heart strugglesto pump against these high pressures. Patients with PAH typicallydevelop significant increases in PVR and sustained elevations in mPAP,which ultimately lead to right ventricular failure and death. Patientsdiagnosed with PAH have a poor prognosis and equally compromised qualityof life, with a mean life expectancy of 2 to 5 years from the time ofdiagnosis if untreated.

A variety of factors contribute to the pathogenesis of pulmonaryhypertension including proliferation of pulmonary cells which cancontribute to vascular remodeling (i.e., hyperplasia). For example,pulmonary vascular remodeling occurs primarily by proliferation ofarterial endothelial cells and smooth muscle cells of patients withpulmonary hypertension. Overexpression of various cytokines is believedto promote pulmonary hypertension. Further, it has been found thatpulmonary hypertension may rise from the hyperproliferation of pulmonaryarterial smooth cells and pulmonary endothelial cells. Still further,advanced PAH may be characterized by muscularization of distal pulmonaryarterioles, concentric intimal thickening, and obstruction of thevascular lumen by proliferating endothelial cells. Pietra et al., J. Am.Coll. Cardiol., 43:255-325 (2004).

PAH can be diagnosed based on a mean pulmonary artery pressure of above25 mmHg (or above 20 mmHg under updated guidelines) at rest, with anormal pulmonary artery capillary wedge pressure. PAH can lead toshortness of breath, dizziness, fainting, and other symptoms, all ofwhich are exacerbated by exertion. PAH can be a severe disease with amarkedly decreased exercise tolerance and heart failure. Two major typesof PAH include idiopathic PAH (e.g., PAH in which no predisposing factoris identified) and heritable PAH (e.g., PAH associated with a mutationin BMPR2, ALK1, ENG, SMAD9, CAV1, KCNK3, or EIF2AK4). In 70% of familialPAH cases, mutations are located in the BMPR2 gene. Risk factors for thedevelopment of PAH include family history of PAH, drug and toxin use(e.g., methamphetamine or cocaine use), infection (e.g., HIV infectionor schistosomiasis), cirrhosis of the liver, congenital heartabnormalities, portal hypertension, pulmonary veno-occlusive disease,pulmonary capillary hemangiomatosis, or connective tissue/autoimmunedisorders (e.g., scleroderma or lupus). PAH may be associated with longterm responders to calcium channel blockers, overt features ofvenous/capillaries (PVOD/PCH) involvement, and persistent PH of thenewborn syndrome.

Group 2 PH

Pulmonary hypertension due to left heart disease (PH-LHD) (WHO Group 2PH) is a complex pathophenotype that, when present, may result in anincreased susceptibility to adverse events and worse clinical outcome.PH-LHD is sometimes defined as patients having a pulmonary capillarywedge pressure (PCWP)>15 mmHg and a mean pulmonary artery pressure(mPAP)≥25 mmHg (or a mean pulmonary artery pressure (mPAP)≥20 mmHg underupdated guidelines). PH-LHD occurs as a consequence of the backwardtransmission of high left sided filling pressures, mainly driven by LVdiastolic function, directly to the post-capillary pulmonary vesselsand, thereby, to the rest of the pulmonary circulation. PH-LHD may beassociated with or caused by PH due to heart failure with preserved leftventricle ejection fraction (LVEF) [also known as HFpEF], PH due toheart failure with reduced LVEF (also known as HFrEF), valvular heartdisease (VHD), or congenital/acquired cardiovascular conditions leadingto post-capillary PH. Compared with PAH, patients with PH-LHD are oftenolder, female, with a higher prevalence of cardiovascular co-morbiditiesand most, if not all, of the features of metabolic syndrome.

Valvular heart disease (VHD) associated with pulmonary hypertension mayresult from multiple mechanisms such as an increase in PVR, pulmonaryblood flow, or pulmonary venous pressure. The chronic rise in PAPfrequently leads to RV pressure overload and subsequent RV failure.Clinical signs and symptoms of left-sided VHD with PH are orthopnea andparoxysmal nocturnal dyspnea. In advanced stages of diseases, signs ofRV failure including peripheral edema, ascites, and syncope arefrequently observed. There are four valvular heart disease subtypeswhich include mitral valve stenosis, mitral valve regurgitation, aorticstenosis, and aortic regurgitation.

Mitral valve stenosis occurs when the heart's mitral valve is narroweddue to the valve becoming stiff or scarred, or the valve flaps partiallyjoining together. This results in the valve not opening as widely as itshould, which causes poor blood flow and may result in blood backing upinto the lungs. Left untreated, mitral valve stenosis can lead toserious heart complications. Common causes of mitral valve stenosisinclude rheumatic heart disease, radiation, and mitral annuluscalcification. Typical interventions for mitral stenosis include balloonvavuloplasty, commisurrotomy, and surgical valve replacement.

Mitral valve regurgitation (also called mitral insufficiency) occurswhen the flaps (leaflets) of the mitral valve do not close tightly,allowing blood to flow backward in the heart. As a result, blood can'tmove through the heart or to the rest of the body as efficiently,resulting in fatigue or shortness of breath. Additionally, the reducedflow increases pressure in the left atrium and lung vasculature. Inmoderate to severe cases, surgery may be recommended to either repair orreplace the damaged valve. Left untreated, severe mitral valveregurgitation can cause heart failure or serious heart rhythm problems.Common causes of mitral valve regurgitation include degenerative mitraldisease such as mitral valve prolapse and mitral annulus calcification.Typical interventions for mitral valve regurgitation includetranscatheter mitral valve repair, surgical repair, or replacement.

In aortic stenosis, the aortic valve does not open fully. This decreasesblood flow from the heart. As the aortic valve becomes more narrow, thepressure increases inside the left heart ventricle. This causes the leftheart ventricle to become thicker, which decreases blood flow and canlead to chest pain. As the pressure continues to rise, blood may back upinto the lungs causing dyspnea. Severe forms of aortic stenosis preventenough blood from reaching the brain and rest of the body. Common causesof aortic stenosis include calcification of the aortic valve or thepresence of a bicuspid aortic valve. Typical interventions includetranscatheter aortic valve replacement (percutaneous valve replacement)and surgical valve replacement.

Aortic regurgitation (also known as aortic insufficiency) occurs whenthe aortic valve is unable to fully close. The valve leaks, resulting inreduced blood flow. As a result, the heart has to work harder to make upfor the reduced blood flow, and over time it will weaken. Because ofthis, the amount of blood that flows from the heart to the rest of thebody is reduced. Common causes of aortic regurgitation include aorticroot dilatation and presence of a bicuspid aortic valve.

Among those patients with PH-LHD, two phenotypes have been described: 1)a group of isolated post-capillary (IpcPH) or “passive” PH in whichelevated pulmonary pressures are reversible and in proportion toincreases in left atrial pressure, and 2) a group with an added“pre-capillary” component [combined post-capillary and pre-capillary PH(CpcPH)]. This latter group, CpcPH, may have comorbid pulmonary vascularremodeling and therefore may demonstrate persistent PH afterinterventions to lower left sided filling pressures.

In some embodiments, a combination of mPAP, PAWP, PVR, or DPG may beused to define the different subtypes of PH-LHD, i.e., IpcPH and CpcPH(see, e.g., Table 2). In some embodiments, patients with CpcPH arecharacterized as having a TPG >12-15 mmHg and a PVR >2.5-3 Wood units(WU). In some embodiments, CpcPH is distinguished from IpcPH using theDPG. In some embodiments, a patient with CpcPH has a DPG ≥7 mmHg. Insome embodiments, a patient with IpcPH has a DPG <7 mmHg.

In some embodiments, a combination of DPG and PVR may be used to definethe different types of PH-LHD. For instance, in some embodiments, IpcPHpatients have a DPG <7 mmHg and/or a PVR of ≤3 WU. In some embodiments,CpcPH patients have a DPG ≥7 mmHg and/or a PVR >3 WU.

The clinical classification or hematological classification describedherein and the associated diagnostic parameters may be updated when newdata are available or when additional clinical entities are considered.For instance, at the 5^(th) World Symposium on Pulmonary Hypertension(WSPH), a new terminology was adopted to distinguish IpcPH from CpcPH,based on the diastolic pressure difference/gradient (DPG) between thedPAP and PAWP. However, this definition was found to be too restrictiveand exposed to interpretation, leading to controversies about whetherthe DPG would or would not predict outcome in patients with group 2 PH.Accordingly, at the 6^(th) WSPH, pulmonary vascular resistance (PVR) wassubsequently reintroduced to better reflect the impact of the rightventricle on patient outcome. See, e.g., Vachiery J. L., et al. EurRespir J 2019 Jan. 24; 53(1).

Therapies for treating PH-LHD primarily include treatment of theunderlying condition (i.e., COPD, sleep apnea syndrome, CTEPH) prior toconsidering specific measures to treat the PH itself. Some therapiesinclude repair of valvular heart disease (if indicated). Non-specificvasodilators such as nitrates and hydralazine may also be used. In someembodiments, an LV assist device (LVAD) may be used to lower pulmonarypressure. The lack of specific therapies is particularly problematicbecause PH-LHD is the most common cause of PH in western countries andits presence commonly results in adverse course of the disease.Specifically, the presence of PH-LHD can result in more severe symptomsin LHD, worse exercise tolerance, and a negative impact on outcome.

Group 3 PH

Pulmonary hypertension due to lung disease and/or hypoxia (WHO Group 3PH) refers to a form of pulmonary hypertension that is due to lungdisease or chronic hypoxia. This form of PH is also known as “hypoxicPH” or “hypoxic pulmonary hypertension.” Hypoxic PH may be associatedwith or caused by chronic obstructive pulmonary disease (e.g.,emphysema), interstitial lung disease, sleep-disordered breathing (e.g.,sleep apnea), lung disease (e.g., pulmonary fibrosis), alveolarhypoventilation disorders, chronic exposure to high altitude, ordevelopmental abnormalities.

Group 4 PH

Pulmonary hypertension due to pulmonary artery obstructions (WHO Group 4PH) is a form of pulmonary hypertension that is related to chronicarterial obstruction (e.g., blood clots). There may be multiplepathophysiological mechanisms driving development of PH in Group 4including chronic thromboembolic PH, sarcoma (high or intermediategrade) or angiosarcoma, other malignant tumors (e.g., renal carcinoma,uterine carcinoma, germ cell tumors of the testis, or other tumors),non-malignant tumors (e.g., uterine leiomyoma), arteritis withoutconnective tissue disease, congenital pulmonary artery stenosis, orparasites (e.g., hydatidosis).

Various pulmonary hemodynamic parameters are associated with Group 4 PH.For instance, in patients with PH due to pulmonary artery obstructions,those with severe PH (>40 mmHg) often have a marked increase in PVR(around 10 WU); more often these patients may have a mild PH (mPAP 20-30mmHg), associated with lower PVR but remaining generally >3 WU. See,e.g., Simonneau (2019) Eur Respir J: 53:1801913. In these differentchronic lung diseases, even a modest elevation in mPAP (20-29 mmHg) canbe associated with a poor prognosis. Furthermore, in chronicthromboembolism, patients may have severe pre-capillary PH with a mPAPof about 47 mmHg and a mean PVR of about 8.9 WU. Id. In this setting,even in patients with mild elevation of mPAP (20-24 mmHg), PVR isgenerally >3 WU.

Group 5 PH

Pulmonary hypertension with unclear and/or multifactorial mechanisms(WHO Group 5 PH) is a group which contains less-studied forms of PH incomparison with the other groups. However, many of the PH formscurrently in group 5 represent a significant part of the PH burden. Thediseases within Group 5 PH are characterized by having no identifiedpredominant mechanism driving the development of PH. There may bemultiple pathophysiological mechanisms driving development of PH,including hematological disorders (e.g., chronic hemolytic anemia ormyeloproliferative disorders), systemic and metabolic disorders (e.g.,Pulmonary Langerhans cell histiocytosis, Gaucher disease, glycogenstorage disease, neurofibromatosis, or sarcoidosis), others (e.g.,chronic renal failure with or without hemodialysis or fibrosingmediastinitis), or complex congenital heart disease.

Measurements of PH

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of post-capillary pulmonaryhypertension in WHO Group 2 and/or Group 5 PH) comprising administeringto a patient in need thereof an effective amount of an ActRIIpolypeptide (e.g., an amino acid sequence that is at least 90% identicalto an amino acid sequence corresponding to residues 30-110 of SEQ ID NO:1). In some embodiments, the method relates to treating PcPH patientsthat have IpcPH. In some embodiments, the method relates to treatingPcPH patients that have CpcPH. In some embodiments, the method relatesto treating PcPH patients that have pulmonary hypertension due to leftheart disease (PH-LHD). In some embodiments, the method relates totreating PcPH patients that have Group 2 PH as classified by the WHO. Insome embodiments, the method relates to treating PcPH patients that havepulmonary hypertension due to heart failure with preserved LVEF (HFpEF).In some embodiments, the method relates to treating PcPH patients thathave pulmonary hypertension due to heart failure with reduced LVEF(HFrEF). In some embodiments, the method relates to treating PcPHpatients that have valvular heart disease. In some embodiments, thevalvular heart disease is aortic regurgitation. In some embodiments, thevalvular heart disease is aortic stenosis. In some embodiments, thevalvular heart disease is mitral valve disease. In some embodiments, thevalvular heart disease is mitral valve regurgitation. In someembodiments, the valvular heart disease is mitral valve stenosis. Insome embodiments, the method relates to treating CpcPH patients who havePH due to valvular heart disease. In some embodiments, the methodrelates to treating IpcPH patients who have PH due to valvular heartdisease. In some embodiments, the method relates to treating PcPHpatients that have congenital/acquired cardiovascular conditions leadingto post-capillary PH. In some embodiments, the method relates totreating PcPH patients that have pulmonary hypertension with unclearand/or multifactorial mechanisms. In some embodiments, the methodrelates to treating PcPH patients that have Group 5 PH as classified bythe WHO.

In some embodiments, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity ofpulmonary hypertension in combinations of certain patient populations.Each of the patient populations described herein can be combined andreorganized accordingly. For instance, in some embodiments, the methodrelates to treating CpcPH patients who have PH due to heart failure withpreserved LVEF (HFpEF). In some embodiments, the method relates totreating CpcPH patients who have PH due to heart failure with reducedLVEF (HFrEF). In some embodiments, the method relates to treating CpcPHpatients who have PH due to valvular heart disease. In some embodiments,the method relates to treating IpcPH patients who have PH due to heartfailure with preserved LVEF (HFpEF). In some embodiments, the methodrelates to treating IpcPH patients who have PH due to heart failure withreduced LVEF (HFrEF). In some embodiments, the method relates totreating IpcPH patients who have PH due to valvular heart disease.

In some embodiments, the method relates to pulmonary hypertensionpatients that have pulmonary hypertension with unclear and/ormultifactorial mechanisms. In some embodiments, the method relates topatients that have a hematological disorder (e.g., chronic hemolyticanemia and myeloproliferative disorders). In some embodiments, themethod relates to patients that have a systemic and/or metabolicdisorder (e.g., pulmonary langerhans cell histiocytosis, Gaucherdisease, glycogen storage disease, neurofibromatosis, and sarcoidosis).

In some embodiments, the method relates to pulmonary hypertensionpatients that have other disorders with unclear and/or multifactorialmechanisms (e.g., chronic renal failure with or without hemodialysis orfibrosing mediastinitis). In some embodiments, the method relates topatients that have complex congenital heart disease.

mPAP

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the patient has restingmean pulmonary arterial pressure (mPAP) of at least 20 mmHg (e.g., 20,25, 30, 35, 40, 45, or 50 mmHg). As used herein, the terms “meanpulmonary arterial pressure” and mean pulmonary artery pressure are usedinterchangeably. In some embodiments, the method relates to patientshaving a resting mPAP of at least 20 mmHg. In some embodiments, themethod relates to patients having a resting mPAP of at least 25 mmHg. Insome embodiments, the method relates to patients having a resting mPAPof at least 30 mmHg. In some embodiments, the method relates to patientshaving a resting mPAP of at least 35 mmHg. In some embodiments, themethod relates to patients having a resting mPAP of at least 40 mmHg. Insome embodiments, the method relates to patients having a resting mPAPof at least 45 mmHg. In some embodiments, the method relates to patientshaving a resting mPAP of at least 50 mmHg.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates toimproving the pulmonary arterial pressure in the patient. In someembodiments, the improvement in pulmonary arterial pressure is areduction in the mean pulmonary arterial pressure (mPAP). In someembodiments, the method relates to reducing mPAP. In some embodiments,the method relates to reducing the patient's mPAP by at least 1 mmHg. Insome embodiments, the method relates to reducing the patient's mPAP byat least 2 mmHg. In some embodiments, the method relates to reducing thepatient's mPAP by at least 3 mmHg. In certain embodiments, the methodrelates to reducing the patient's mPAP by at least 5 mmHg. In certainembodiments, the method relates to reducing the patient's mPAP by atleast 7 mmHg. In certain embodiments, the method relates to reducing thepatient's mPAP by at least 10 mmHg. In certain embodiments, the methodrelates to reducing the patient's mPAP by at least 12 mmHg. In certainembodiments, the method relates to reducing the patient's mPAP by atleast 15 mmHg. In certain embodiments, the method relates to reducingthe patient's mPAP by at least 20 mmHg. In certain embodiments, themethod relates to reducing the patient's mPAP by at least 25 mmHg.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates todecreasing the patient's mPAP by least 1% (e.g., 1, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). Insome embodiments, the method relates to decreasing the patient's mPAP byat least 1%. In some embodiments, the method relates to decreasing thepatient's mPAP by at least 5%. In some embodiments, the method relatesto decreasing the patient's mPAP by at least 10%. In some embodiments,the method relates to decreasing the patient's mPAP by at least 15%. Insome embodiments, the method relates to decreasing the patient's mPAP byat least 20%. In some embodiments, the method relates to decreasing thepatient's mPAP by at least 25%. In some embodiments, the method relatesto decreasing the patient's mPAP by at least 30%. In some embodiments,the method relates to decreasing the patient's mPAP by at least 35%. Insome embodiments, the method relates to decreasing the patient's mPAP byat least 40%. In some embodiments, the method relates to decreasing thepatient's mPAP by at least 45%. In some embodiments, the method relatesto decreasing the patient's mPAP by at least 50%. In some embodiments,the method relates to decreasing the patient's mPAP by at least 55%. Insome embodiments, the method relates to decreasing the patient's mPAP byat least 60%. In some embodiments, the method relates to decreasing thepatient's mPAP by at least 65%. In some embodiments, the method relatesto decreasing the patient's mPAP by at least 70%. In some embodiments,the method relates to decreasing the patient's mPAP by at least 75%. Insome embodiments, the method relates to decreasing the patient's mPAP byat least 80%. In some embodiments, the method relates to decreasing thepatient's mPAP by at least 85%. In some embodiments, the method relatesto decreasing the patient's mPAP by at least 90%. In some embodiments,the method relates to decreasing the patient's mPAP by at least 95%. Insome embodiments, the method relates to decreasing the patient's mPAP byat least 100%.

mRAP

In some patients, increased pulmonary vascular resistance to blood flowleads to increased right atrial pressure (RAP) and right heart failure.Patients with right heart failure typically have an increased ratio ofRAP and pulmonary artery wedge pressure (PAWP). In certain aspects, thedisclosure relates to methods of treating, preventing, or reducing theprogression rate and/or severity of PcPH (e.g., treating, preventing, orreducing the progression rate and/or severity of one or morecomplications of PcPH in WHO Group 2 and/or Group 5 PH) comprisingadministering to a patient in need thereof an effective amount of anActRII polypeptide (e.g., an amino acid sequence that is at least 90%identical to an amino acid sequence corresponding to residues 30-110 ofSEQ ID NO: 1), wherein the patient has resting mean right atrialpressure (mRAP) of at least 5 mmHg (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 18, 20, 21, 22, 23, 24, or 25 mmHg). In someembodiments, the method relates to a patient having a resting mRAP of atleast 5 mmHg. In some embodiments, the method relates to a patienthaving a resting mRAP of at least 6 mmHg. In some embodiments, themethod relates to a patient having a resting mRAP of at least 7 mmHg. Insome embodiments, the method relates to a patient having a resting mRAPof at least 8 mmHg. In some embodiments, the method relates to a patienthaving a resting mRAP of at least 9 mmHg. In some embodiments, themethod relates to a patient having a resting mRAP of at least 10 mmHg.In some embodiments, the method relates to a patient having a restingmRAP of at least 11 mmHg. In some embodiments, the method relates to apatient having a resting mRAP of at least 12 mmHg. In some embodiments,the method relates to a patient having a resting mRAP of at least 13mmHg. In some embodiments, the method relates to a patient having aresting mRAP of at least 14 mmHg. In some embodiments, the methodrelates to a patient having a resting mRAP of at least 15 mmHg. In someembodiments, the method relates to a patient having a resting mRAP of atleast 16 mmHg. In some embodiments, the method relates to a patienthaving a resting mRAP of at least 17 mmHg. In some embodiments, themethod relates to a patient having a resting mRAP of at least 18 mmHg.In some embodiments, the method relates to a patient having a restingmRAP of at least 19 mmHg. In some embodiments, the method relates to apatient having a resting mRAP of at least 20 mmHg. In some embodiments,the method relates to a patient having a resting mRAP of at least 21mmHg. In some embodiments, the method relates to a patient having aresting mRAP of at least 22 mmHg. In some embodiments, the methodrelates to a patient having a resting mRAP of at least 23 mmHg. In someembodiments, the method relates to a patient having a resting mRAP of atleast 24 mmHg. In some embodiments, the method relates to a patienthaving a resting mRAP of at least 25 mmHg.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates toimproving the mean right atrial pressure in the patient. In someembodiments, the improvement in the mean right atrial pressure (mRAP) isa reduction in the mRAP. In some embodiments, the method relates toreducing mRAP. In some embodiments, the method relates to reducing thepatient's mRAP by at least 1 mmHg. In some embodiments, the methodrelates to reducing the patient's mRAP by at least 2 mmHg. In someembodiments, the method relates to reducing the patient's mRAP by atleast 3 mmHg. In some embodiments, the method relates to reducing thepatient's mRAP by at least 4 mmHg. In certain embodiments, the methodrelates to reducing the patient's mRAP by at least 5 mmHg. In someembodiments, the method relates to reducing the patient's mRAP by atleast 6 mmHg. In certain embodiments, the method relates to reducing thepatient's mRAP by at least 7 mmHg. In some embodiments, the methodrelates to reducing the patient's mRAP by at least 8 mmHg. In someembodiments, the method relates to reducing the patient's mRAP by atleast 9 mmHg. In certain embodiments, the method relates to reducing thepatient's mRAP by at least 10 mmHg. In some embodiments, the methodrelates to reducing the patient's mRAP by at least 11 mmHg. In certainembodiments, the method relates to reducing the patient's mRAP by atleast 12 mmHg. In some embodiments, the method relates to reducing thepatient's mRAP by at least 13 mmHg. In some embodiments, the methodrelates to reducing the patient's mRAP by at least 14 mmHg. In certainembodiments, the method relates to reducing the patient's mRAP by atleast 15 mmHg. In some embodiments, the method relates to reducing thepatient's mRAP by at least 16 mmHg. In some embodiments, the methodrelates to reducing the patient's mRAP by at least 17 mmHg. In someembodiments, the method relates to reducing the patient's mRAP by atleast 18 mmHg. In some embodiments, the method relates to reducing thepatient's mRAP by at least 19 mmHg. In certain embodiments, the methodrelates to reducing the patient's mRAP by at least 20 mmHg.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates todecreasing the patient's mRAP by least 1% (e.g., 1, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). Insome embodiments, the method relates to decreasing the patient's mRAP byat least 1%. In some embodiments, the method relates to decreasing thepatient's mRAP by at least 5%. In some embodiments, the method relatesto decreasing the patient's mRAP by at least 10%. In some embodiments,the method relates to decreasing the patient's mRAP by at least 15%. Insome embodiments, the method relates to decreasing the patient's mRAP byat least 20%. In some embodiments, the method relates to decreasing thepatient's mRAP by at least 25%. In some embodiments, the method relatesto decreasing the patient's mRAP by at least 30%. In some embodiments,the method relates to decreasing the patient's mRAP by at least 35%. Insome embodiments, the method relates to decreasing the patient's mRAP byat least 40%. In some embodiments, the method relates to decreasing thepatient's mRAP by at least 45%. In some embodiments, the method relatesto decreasing the patient's mRAP by at least 50%. In some embodiments,the method relates to decreasing the patient's mRAP by at least 55%. Insome embodiments, the method relates to decreasing the patient's mRAP byat least 60%. In some embodiments, the method relates to decreasing thepatient's mRAP by at least 65%. In some embodiments, the method relatesto decreasing the patient's mRAP by at least 70%. In some embodiments,the method relates to decreasing the patient's mRAP by at least 75%. Insome embodiments, the method relates to decreasing the patient's mRAP byat least 80%. In some embodiments, the method relates to decreasing thepatient's mRAP by at least 85%. In some embodiments, the method relatesto decreasing the patient's mRAP by at least 90%. In some embodiments,the method relates to decreasing the patient's mRAP by at least 95%. Insome embodiments, the method relates to decreasing the patient's mRAP byat least 100%.

PVR

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the patient has a pulmonaryvascular resistance (PVR) of at least 2.5 Woods Units (e.g., 2.5, 3, 4,5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 Woods Units). In someembodiments, the method relates to patients having a PVR of at least 2.5Woods Units. In some embodiments, the method relates to patients havinga PVR of at least 3 Woods Units. In some embodiments, the method relatesto patients having a PVR of at least 4 Woods Units. In some embodiments,the method relates to patients having a PVR of at least 5 Woods Units.In some embodiments, the method relates to patients having a PVR of atleast 6 Woods Units. In some embodiments, the method relates to patientshaving a PVR of at least 7 Woods Units. In some embodiments, the methodrelates to patients having a PVR of at least 8 Woods Units. In someembodiments, the method relates to patients having a PVR of at least 9Woods Units. In some embodiments, the method relates to patients havinga PVR of at least 10 Woods Units. In some embodiments, the methodrelates to patients having a PVR of at least 12 Woods Units. In someembodiments, the method relates to patients having a PVR of at least 14Woods Units. In some embodiments, the method relates to patients havinga PVR of at least 16 Woods Units. In some embodiments, the methodrelates to patients having a PVR of at least 18 Woods Units. In someembodiments, the method relates to patients having a PVR of at least 20Woods Units.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates toreducing the patient's PVR. In some embodiments, the reduction in thepatient's PVR is a result of a decrease in the patient's mean pulmonaryarterial pressure (mPAP). In some embodiments, the method relates toreducing the patient's PVR by at least 0.5 Wood Units. In someembodiments, the method relates to reducing the patient's PVR by atleast 1 Wood Units. In some embodiments, the method relates to reducingthe patient's PVR by at least 2 Wood Units. In some embodiments, themethod relates to reducing the patient's PVR by at least 4 Wood Units.In some embodiments, the method relates to reducing the patient's PVR byat least 6 Wood Units. In some embodiments, the method relates toreducing the patient's PVR by at least 8 Wood Units. In someembodiments, the method relates to reducing the patient's PVR by atleast 10 Wood Units.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates todecreasing the patient's PVR. In some embodiments, the reduction in thepatient's PVR is a result of a decrease in the patient's mean pulmonaryarterial pressure (mPAP). In some embodiments, the method relates todecreasing the patient's PVR by least 1% (e.g., 1%, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). Insome embodiments, the method relates to decreasing the patient's PVR byat least 1%. In some embodiments, the method relates to decreasing thepatient's PVR by at least 5%. In some embodiments, the method relates todecreasing the patient's PVR by at least 10%. In some embodiments, themethod relates to decreasing the patient's PVR by at least 15%. In someembodiments, the method relates to decreasing the patient's PVR by atleast 20%. In some embodiments, the method relates to decreasing thepatient's PVR by at least 25%. In some embodiments, the method relatesto decreasing the patient's PVR by at least 30%. In some embodiments,the method relates to decreasing the patient's PVR by at least 35%. Insome embodiments, the method relates to decreasing the patient's PVR byat least 40%. In some embodiments, the method relates to decreasing thepatient's PVR by at least 45%. In some embodiments, the method relatesto decreasing the patient's PVR by at least 50%. In some embodiments,the method relates to decreasing the patient's PVR by at least 55%. Insome embodiments, the method relates to decreasing the patient's PVR byat least 60%. In some embodiments, the method relates to decreasing thepatient's PVR by at least 65%. In some embodiments, the method relatesto decreasing the patient's PVR by at least 70%. In some embodiments,the method relates to decreasing the patient's PVR by at least 75%. Insome embodiments, the method relates to decreasing the patient's PVR byat least 80%. In some embodiments, the method relates to decreasing thepatient's PVR by at least 85%. In some embodiments, the method relatesto decreasing the patient's PVR by at least 90%. In some embodiments,the method relates to decreasing the patient's PVR by at least 95%. Insome embodiments, the method relates to decreasing the patient's PVR byat least 100%.

PAWP

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH WHO Group 2 and/or Group 5PH) comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1), wherein the patient has pulmonary arterialwedge pressure (PAWP) of at least 12 mmHg (e.g., 12, 15, 20, 25, 30, 35,40, 45, or 50 mmHg). In some embodiments, the method relates to patientshaving a PAWP of at least 15 mmHg. In some embodiments, the methodrelates to patients having a PAWP of at least 20 mmHg. In someembodiments, the method relates to patients having a PAWP of at least 25mmHg. In some embodiments, the method relates to patients having a PAWPof at least 30 mmHg. In some embodiments, the method relates to patientshaving a PAWP of at least 35 mmHg. In some embodiments, the methodrelates to patients having a PAWP of at least 40 mmHg. In someembodiments, the method relates to patients having a PAWP of at least 45mmHg. In some embodiments, the method relates to patients having a PAWPof at least 50 mmHg. In some embodiments, the method relates to patientshaving a PCWP between 15 to 30 mmHg.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates toreducing the patient's PAWP by at least 1 mmHg. In some embodiments, themethod relates to reducing the patient's PAWP by at least 2 mmHg. Insome embodiments, the method relates to reducing the patient's PAWP byat least 4 mmHg. In some embodiments, the method relates to reducing thepatient's PAWP by at least 6 mmHg. In some embodiments, the methodrelates to reducing the patient's PAWP by at least 10 mmHg. In someembodiments, the method relates to reducing the patient's PAWP by atleast 15 mmHg. In some embodiments, the method relates to reducing thepatient's PAWP by at least 20 mmHg. In some embodiments, the methodrelates to reducing the patient's PAWP by at least 25 mmHg. In someembodiments, the method relates to reducing the patient's PAWP by atleast 30 mmHg.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates todecreasing the patient's PAWP by least 1% (e.g., 1, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). Insome embodiments, the method relates to decreasing the patient's PAWP byat least 1%. In some embodiments, the method relates to decreasing thepatient's PAWP by at least 5%. In some embodiments, the method relatesto decreasing the patient's PAWP by at least 10%. In some embodiments,the method relates to decreasing the patient's PAWP by at least 15%. Insome embodiments, the method relates to decreasing the patient's PAWP byat least 20%. In some embodiments, the method relates to decreasing thepatient's PAWP by at least 25%. In some embodiments, the method relatesto decreasing the patient's PAWP by at least 30%. In some embodiments,the method relates to decreasing the patient's PAWP by at least 35%. Insome embodiments, the method relates to decreasing the patient's PAWP byat least 40%. In some embodiments, the method relates to decreasing thepatient's PAWP by at least 45%. In some embodiments, the method relatesto decreasing the patient's PAWP by at least 50%. In some embodiments,the method relates to decreasing the patient's PAWP by at least 55%. Insome embodiments, the method relates to decreasing the patient's PAWP byat least 60%. In some embodiments, the method relates to decreasing thepatient's PAWP by at least 65%. In some embodiments, the method relatesto decreasing the patient's PAWP by at least 70%. In some embodiments,the method relates to decreasing the patient's PAWP by at least 75%. Insome embodiments, the method relates to decreasing the patient's PAWP byat least 80%. In some embodiments, the method relates to decreasing thepatient's PAWP by at least 85%. In some embodiments, the method relatesto decreasing the patient's PAWP by at least 90%. In some embodiments,the method relates to decreasing the patient's PAWP by at least 95%. Insome embodiments, the method relates to decreasing the patient's PAWP byat least 100%.

LVEDP

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the patient has leftventricular end diastolic pressure (LVEDP) of at least 12 mmHg (e.g.,12, 15, 20, 25, 30, 35, 40, 45, or 50 mmHg). In some embodiments, themethod relates to patients having a LVEDP of at least 15 mmHg. In someembodiments, the method relates to patients having a LVEDP of at least20 mmHg. In some embodiments, the method relates to patients having aLVEDP of at least 25 mmHg. In some embodiments, the method relates topatients having a LVEDP of at least 30 mmHg. In some embodiments, themethod relates to patients having a LVEDP of at least 35 mmHg. In someembodiments, the method relates to patients having a LVEDP of at least40 mmHg. In some embodiments, the method relates to patients having aLVEDP of at least 45 mmHg. In some embodiments, the method relates topatients having a LVEDP of at least 50 mmHg.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates toreducing the patient's LVEDP by at least 1 mmHg. In some embodiments,the method relates to reducing the patient's LVEDP by at least 2 mmHg.In some embodiments, the method relates to reducing the patient's LVEDPby at least 4 mmHg. In some embodiments, the method relates to reducingthe patient's LVEDP by at least 6 mmHg. In some embodiments, the methodrelates to reducing the patient's LVEDP by at least 10 mmHg. In someembodiments, the method relates to reducing the patient's LVEDP by atleast 15 mmHg. In some embodiments, the method relates to reducing thepatient's LVEDP by at least 20 mmHg. In some embodiments, the methodrelates to reducing the patient's LVEDP by at least 25 mmHg. In someembodiments, the method relates to reducing the patient's LVEDP by atleast 30 mmHg.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates todecreasing the patient's LVEDP by least 1% (e.g., 1, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). Insome embodiments, the method relates to decreasing the patient's LVEDPby at least 1%. In some embodiments, the method relates to decreasingthe patient's LVEDP by at least 5%. In some embodiments, the methodrelates to decreasing the patient's LVEDP by at least 10%. In someembodiments, the method relates to decreasing the patient's LVEDP by atleast 15%. In some embodiments, the method relates to decreasing thepatient's LVEDP by at least 20%. In some embodiments, the method relatesto decreasing the patient's LVEDP by at least 25%. In some embodiments,the method relates to decreasing the patient's LVEDP by at least 30%. Insome embodiments, the method relates to decreasing the patient's LVEDPby at least 35%. In some embodiments, the method relates to decreasingthe patient's LVEDP by at least 40%. In some embodiments, the methodrelates to decreasing the patient's LVEDP by at least 45%. In someembodiments, the method relates to decreasing the patient's LVEDP by atleast 50%. In some embodiments, the method relates to decreasing thepatient's LVEDP by at least 55%. In some embodiments, the method relatesto decreasing the patient's LVEDP by at least 60%. In some embodiments,the method relates to decreasing the patient's LVEDP by at least 65%. Insome embodiments, the method relates to decreasing the patient's LVEDPby at least 70%. In some embodiments, the method relates to decreasingthe patient's LVEDP by at least 75%. In some embodiments, the methodrelates to decreasing the patient's LVEDP by at least 80%. In someembodiments, the method relates to decreasing the patient's LVEDP by atleast 85%. In some embodiments, the method relates to decreasing thepatient's LVEDP by at least 90%. In some embodiments, the method relatesto decreasing the patient's LVEDP by at least 95%. In some embodiments,the method relates to decreasing the patient's LVEDP by at least 100%.

DPG

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the patient has restingdiastolic pressure gradient (DPG) of at least 5 mmHg (e.g., 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, or 45 mmHg). In some embodiments, themethod relates to patients having a DPG of at least 5 mmHg. In someembodiments, the method relates to patients having a DPG of at least 6mmHg. In some embodiments, the method relates to patients having a DPGof at least 7 mmHg. In some embodiments, the method relates to patientshaving a DPG of at least 8 mmHg. In some embodiments, the method relatesto patients having a DPG of at least 9 mmHg. In some embodiments, themethod relates to patients having a DPG of at least 10 mmHg. In someembodiments, the method relates to patients having a DPG of at least 15mmHg. In some embodiments, the method relates to patients having a DPGof at least 20 mmHg. In some embodiments, the method relates to patientshaving a DPG of at least 25 mmHg. In some embodiments, the methodrelates to patients having a DPG of at least 30 mmHg. In someembodiments, the method relates to patients having a DPG of at least 35mmHg. In some embodiments, the method relates to patients having a DPGof at least 40 mmHg. In some embodiments, the method relates to patientshaving a DPG of at least 45 mmHg.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates toreducing the patient's DPG by at least 1 mmHg. In some embodiments, themethod relates to reducing the patient's DPG by at least 2 mmHg. In someembodiments, the method relates to reducing the patient's DPG by atleast 4 mmHg. In some embodiments, the method relates to reducing thepatient's DPG by at least 6 mmHg. In some embodiments, the methodrelates to reducing the patient's DPG by at least 10 mmHg. In someembodiments, the method relates to reducing the patient's DPG by atleast 15 mmHg. In some embodiments, the method relates to reducing thepatient's DPG by at least 20 mmHg. In some embodiments, the methodrelates to reducing the patient's DPG by at least 25 mmHg. In someembodiments, the method relates to reducing the patient's DPG by atleast 30 mmHg.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates todecreasing the patient's DPG by least 1% (e.g., 1, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). Insome embodiments, the method relates to decreasing the patient's DPG byat least 1%. In some embodiments, the method relates to decreasing thepatient's DPG by at least 5%. In some embodiments, the method relates todecreasing the patient's DPG by at least 10%. In some embodiments, themethod relates to decreasing the patient's DPG by at least 15%. In someembodiments, the method relates to decreasing the patient's DPG by atleast 20%. In some embodiments, the method relates to decreasing thepatient's DPG by at least 25%. In some embodiments, the method relatesto decreasing the patient's DPG by at least 30%. In some embodiments,the method relates to decreasing the patient's DPG by at least 35%. Insome embodiments, the method relates to decreasing the patient's DPG byat least 40%. In some embodiments, the method relates to decreasing thepatient's DPG by at least 45%. In some embodiments, the method relatesto decreasing the patient's DPG by at least 50%. In some embodiments,the method relates to decreasing the patient's DPG by at least 55%. Insome embodiments, the method relates to decreasing the patient's DPG byat least 60%. In some embodiments, the method relates to decreasing thepatient's DPG by at least 65%. In some embodiments, the method relatesto decreasing the patient's DPG by at least 70%. In some embodiments,the method relates to decreasing the patient's DPG by at least 75%. Insome embodiments, the method relates to decreasing the patient's DPG byat least 80%. In some embodiments, the method relates to decreasing thepatient's DPG by at least 85%. In some embodiments, the method relatesto decreasing the patient's DPG by at least 90%. In some embodiments,the method relates to decreasing the patient's DPG by at least 95%. Insome embodiments, the method relates to decreasing the patient's DPG byat least 100%.

TPG

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the patient has atranspulmonary gradient (TPG) of at least 10 mmHg (e.g., 10, 11, 12, 13,14, 15, 20, 25, 30, 35, 40, 45, or 50 mmHg). In some embodiments, themethod relates to patients having a TPG of at least 10 mmHg. In someembodiments, the method relates to patients having a TPG of at least 11mmHg. In some embodiments, the method relates to patients having a TPGof at least 12 mmHg. In some embodiments, the method relates to patientshaving a TPG of at least 13 mmHg. In some embodiments, the methodrelates to patients having a TPG of at least 14 mmHg. In someembodiments, the method relates to patients having a TPG of at least 15mmHg. In some embodiments, the method relates to patients having a TPGof at least 20 mmHg. In some embodiments, the method relates to patientshaving a TPG of at least 25 mmHg. In some embodiments, the methodrelates to patients having a TPG of at least 30 mmHg. In someembodiments, the method relates to patients having a TPG of at least 35mmHg. In some embodiments, the method relates to patients having a TPGof at least 40 mmHg. In some embodiments, the method relates to patientshaving a TPG of at least 45 mmHg. In some embodiments, the methodrelates to patients having a TPG of at least 50 mmHg.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates toreducing the patient's TPG by at least 1 mmHg. In some embodiments, themethod relates to reducing the patient's TPG by at least 2 mmHg. In someembodiments, the method relates to reducing the patient's TPG by atleast 4 mmHg. In some embodiments, the method relates to reducing thepatient's TPG by at least 6 mmHg. In some embodiments, the methodrelates to reducing the patient's TPG by at least 10 mmHg. In someembodiments, the method relates to reducing the patient's TPG by atleast 15 mmHg. In some embodiments, the method relates to reducing thepatient's TPG by at least 20 mmHg. In some embodiments, the methodrelates to reducing the patient's TPG by at least 25 mmHg. In someembodiments, the method relates to reducing the patient's TPG by atleast 30 mmHg. In some embodiments, the method relates to reducing thepatient's TPG by at least 40 mmHg.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates todecreasing the patient's TPG by least 1% (e.g., 1, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). Insome embodiments, the method relates to decreasing the patient's TPG byat least 1%. In some embodiments, the method relates to decreasing thepatient's TPG by at least 5%. In some embodiments, the method relates todecreasing the patient's TPG by at least 10%. In some embodiments, themethod relates to decreasing the patient's TPG by at least 15%. In someembodiments, the method relates to decreasing the patient's TPG by atleast 20%. In some embodiments, the method relates to decreasing thepatient's TPG by at least 25%. In some embodiments, the method relatesto decreasing the patient's TPG by at least 30%. In some embodiments,the method relates to decreasing the patient's TPG by at least 35%. Insome embodiments, the method relates to decreasing the patient's TPG byat least 40%. In some embodiments, the method relates to decreasing thepatient's TPG by at least 45%. In some embodiments, the method relatesto decreasing the patient's TPG by at least 50%. In some embodiments,the method relates to decreasing the patient's TPG by at least 55%. Insome embodiments, the method relates to decreasing the patient's TPG byat least 60%. In some embodiments, the method relates to decreasing thepatient's TPG by at least 65%. In some embodiments, the method relatesto decreasing the patient's TPG by at least 70%. In some embodiments,the method relates to decreasing the patient's TPG by at least 75%. Insome embodiments, the method relates to decreasing the patient's TPG byat least 80%. In some embodiments, the method relates to decreasing thepatient's TPG by at least 85%. In some embodiments, the method relatesto decreasing the patient's TPG by at least 90%. In some embodiments,the method relates to decreasing the patient's TPG by at least 95%. Insome embodiments, the method relates to decreasing the patient's TPG byat least 100%.

BNP

Both BNP and NT-proBNP are markers of atrial and ventricular distensiondue to increased intracardiac pressure. The New York Heart Association(NYHA) developed a 4-stage functional classification system forcongestive heart failure (CHF) based on the severity of symptoms.Studies have demonstrated that the measured concentrations ofcirculating BNP and NT-proBNP increase with the severity of CHF based onthe NYHA classification. In certain aspects, the disclosure relates tomethods of treating, preventing, or reducing the progression rate and/orseverity of PcPH (e.g., treating, preventing, or reducing theprogression rate and/or severity of one or more complications of PcPH inWHO Group 2 and/or Group 5 PH) comprising administering to a patient inneed thereof an effective amount of an ActRII polypeptide (e.g., anamino acid sequence that is at least 90% identical to an amino acidsequence corresponding to residues 30-110 of SEQ ID NO: 1), wherein thepatient has a brain natriuretic peptide (BNP) level of at least 100pg/mL (e.g., 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 1000,3000, 5000, 10,000, 15,000, or 20,000 pg/mL). In some embodiments, themethod relates to patient's having a BNP level of at least 100 pg/mL. Insome embodiments, the method relates to patient's having a BNP level ofat least 150 pg/mL. In some embodiments, the method relates to patient'shaving a BNP level of at least 200 pg/mL. In some embodiments, themethod relates to patient's having a BNP level of at least 300 pg/mL. Insome embodiments, the method relates to patient's having a BNP level ofat least 400 pg/mL. In some embodiments, the method relates to patient'shaving a BNP level of at least 500 pg/mL. In some embodiments, themethod relates to patient's having a BNP level of at least 600 pg/mL. Insome embodiments, the method relates to patient's having a BNP level ofat least 700 pg/mL. In some embodiments, the method relates to patient'shaving a BNP level of at least 800 pg/mL. In some embodiments, themethod relates to patient's having a BNP level of at least 900 pg/mL. Insome embodiments, the method relates to patient's having a BNP level ofat least 1000 pg/mL. In some embodiments, the method relates topatient's having a BNP level of at least 5000 pg/mL. In someembodiments, the method relates to patient's having a BNP level of atleast 10,000 pg/mL. In some embodiments, the method relates to patient'shaving a BNP level of at least 15,000 pg/mL. In some embodiments, themethod relates to patient's having a BNP level of at least 20,000 pg/mL.In some embodiments, the method relates to treatment of a patient whohas elevated BNP levels as compared to a healthy patient.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates toreducing the patient's BNP levels by at least 10 pg/mL. In someembodiments, the method relates to reducing the patient's BNP levels byat least 50 pg/mL. In some embodiments, the method relates to reducingthe patient's BNP levels by at least 100 pg/mL. In some embodiments, themethod relates to reducing the patient's BNP levels by at least 200pg/mL. In some embodiments, the method relates to reducing the patient'sBNP levels by at least 300 pg/mL. In some embodiments, the methodrelates to reducing the patient's BNP levels by at least 400 pg/mL. Insome embodiments, the method relates to reducing the patient's BNPlevels by at least 500 pg/mL. In some embodiments, the method relates toreducing the patient's BNP levels by at least 600 pg/mL. In someembodiments, the method relates to reducing the patient's BNP levels byat least 700 pg/mL. In some embodiments, the method relates to reducingthe patient's BNP levels by at least 800 pg/mL. In some embodiments, themethod relates to reducing the patient's BNP levels by at least 900pg/mL. In some embodiments, the method relates to reducing the patient'sBNP levels by at least 1000 pg/mL. In some embodiments, the methodrelates to reducing the patient's BNP levels by at least 5000 pg/mL. Insome embodiments, the method relates to reducing the patient's BNPlevels to normal levels. In some embodiments, normal levels correspondto levels of <100 pg/mL.

In some embodiments, the method relates to reducing the patient's BNP byat least 5% (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100%). In some embodiments, the methodrelates to reducing the patient's BNP by at least 5%. In someembodiments, the method relates to reducing the patient's BNP by atleast 10%. In some embodiments, the method relates to reducing thepatient's BNP by at least 15%. In some embodiments, the method relatesto reducing the patient's BNP by at least 20%. In some embodiments, themethod relates to reducing the patient's BNP by at least 25%. In someembodiments, the method relates to reducing the patient's BNP by atleast 30%. In some embodiments, the method relates to reducing thepatient's BNP by at least 35%. In some embodiments, the method relatesto reducing the patient's BNP by at least 40%. In some embodiments, themethod relates to reducing the patient's BNP by at least 45%. In someembodiments, the method relates to reducing the patient's BNP by atleast 50%. In some embodiments, the method relates to reducing thepatient's BNP by at least 55%. In some embodiments, the method relatesto reducing the patient's BNP by at least 60%. In some embodiments, themethod relates to reducing the patient's BNP by at least 65%. In someembodiments, the method relates to reducing the patient's BNP by atleast 70%. In some embodiments, the method relates to reducing thepatient's BNP by at least 75%. In some embodiments, the method relatesto reducing the patient's BNP by at least 80%. In some embodiments, themethod relates to reducing the patient's BNP by at least 85%. In someembodiments, the method relates to reducing the patient's BNP by atleast 90%. In some embodiments, the method relates to reducing thepatient's BNP by at least 95%. In some embodiments, the method relatesto reducing the patient's BNP by at least 100%.

NT-ProBNP

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the patient has a NT-proBNPlevel of at least 100 pg/mL (e.g., 100, 150, 200, 300, 400, 500, 600,700, 800, 900, 1000, 3000, 5000, 10,000, 15,000, 20,000, 25,000, or30,000 pg/mL). In some embodiments, the method relates to patient'shaving a NT-proBNP level of at least 100 pg/mL. In some embodiments, themethod relates to patient's having a NT-proBNP level of at least 150pg/mL. In some embodiments, the method relates to patient's having aNT-proBNP level of at least 200 pg/mL. In some embodiments, the methodrelates to patient's having a NT-proBNP level of at least 300 pg/mL. Insome embodiments, the method relates to patient's having a NT-proBNPlevel of at least 400 pg/mL. In some embodiments, the method relates topatient's having a NT-proBNP level of at least 500 pg/mL. In someembodiments, the method relates to patient's having a NT-proBNP level ofat least 600 pg/mL. In some embodiments, the method relates to patient'shaving a NT-proBNP level of at least 700 pg/mL. In some embodiments, themethod relates to patient's having a NT-proBNP level of at least 800pg/mL. In some embodiments, the method relates to patient's having aNT-proBNP level of at least 900 pg/mL. In some embodiments, the methodrelates to patient's having a NT-proBNP level of at least 1000 pg/mL. Insome embodiments, the method relates to patient's having a NT-proBNPlevel of at least 5000 pg/mL. In some embodiments, the method relates topatient's having a NT-proBNP level of at least 10,000 pg/mL. In someembodiments, the method relates to patient's having a NT-proBNP level ofat least 15,000 pg/mL. In some embodiments, the method relates topatient's having a NT-proBNP level of at least 20,000 pg/mL. In someembodiments, the method relates to patient's having a NT-proBNP level ofat least 25,000 pg/mL. In some embodiments, the method relates topatient's having a NT-proBNP level of at least 30,000 pg/mL. In someembodiments, the method relates to treatment of a patient who haselevated NT-proBNP levels as compared to a healthy patient.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates toreducing the patient's NT-proBNP levels. In some embodiments, the methodrelates to reducing the patient's NT-proBNP by at least 10 pg/mL. Insome embodiments, the method relates to reducing the patient's NT-proBNPby at least 50 pg/mL. In some embodiments, the method relates toreducing the patient's NT-proBNP by at least 100 pg/mL. In someembodiments, the method relates to reducing the patient's NT-proBNP byat least 200 pg/mL. In some embodiments, the method relates to reducingthe patient's NT-proBNP by at least 300 pg/mL. In some embodiments, themethod relates to reducing the patient's NT-proBNP by at least 400pg/mL. In some embodiments, the method relates to reducing the patient'sNT-proBNP by at least 500 pg/mL. In some embodiments, the method relatesto reducing the patient's NT-proBNP by at least 600 pg/mL. In someembodiments, the method relates to reducing the patient's NT-proBNP byat least 700 pg/mL. In some embodiments, the method relates to reducingthe patient's NT-proBNP by at least 800 pg/mL. In some embodiments, themethod relates to reducing the patient's NT-proBNP by at least 900pg/mL. In some embodiments, the method relates to reducing the patient'sNT-proBNP by at least 1000 pg/mL. In some embodiments, the methodrelates to reducing the patient's NT-proBNP by at least 5000 pg/mL. Insome embodiments, the method relates to reducing the patient's NT-proBNPby at least 10,000 pg/mL. In some embodiments, the method relates toreducing the patient's NT-proBNP by at least 15,000 pg/mL. In someembodiments, the method relates to reducing the patient's NT-proBNP byat least 20,000 pg/mL. In some embodiments, the method relates toreducing the patient's NT-proBNP by at least 25,000 pg/mL.

In some embodiments, the method relates to decreasing the patient'sNT-proBNP levels to a normal level and maintaining their normalNT-proBNP levels. In some embodiments, the disclosure relates to methodsof maintaining one or more hemodynamic parameters in the PcPH patient ata normal level (e.g., normal as compared to healthy people of similarage and sex), comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1). In some embodiments, the methodrelates to maintaining the patient's NT-proBNP levels at a normal level.In some embodiments, the method relates to maintaining the patient'sNT-proBNP level at less than 100 pg/mL. In some embodiments, the methodrelates to maintaining the patient's NT-proBNP level at less than 200pg/mL. In some embodiments, the method relates to maintaining thepatient's NT-proBNP level at less than 300 pg/mL. In some embodiments,the method relates to maintaining the patient's NT-proBNP level at lessthan 400 pg/mL.

In some embodiments, the method relates to reducing the patient'sNT-proBNP by at least 5% (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). In some embodiments, themethod relates to reducing the patient's NT-proBNP by at least 5%. Insome embodiments, the method relates to reducing the patient's NT-proBNPby at least 10%. In some embodiments, the method relates to reducing thepatient's NT-proBNP by at least 15%. In some embodiments, the methodrelates to reducing the patient's NT-proBNP by at least 20%. In someembodiments, the method relates to reducing the patient's NT-proBNP byat least 25%. In some embodiments, the method relates to reducing thepatient's NT-proBNP by at least 30%. In some embodiments, the methodrelates to reducing the patient's NT-proBNP by at least 35%. In someembodiments, the method relates to reducing the patient's NT-proBNP byat least 40%. In some embodiments, the method relates to reducing thepatient's NT-proBNP by at least 45%. In some embodiments, the methodrelates to reducing the patient's NT-proBNP by at least 50%. In someembodiments, the method relates to reducing the patient's NT-proBNP byat least 55%. In some embodiments, the method relates to reducing thepatient's NT-proBNP by at least 60%. In some embodiments, the methodrelates to reducing the patient's NT-proBNP by at least 65%. In someembodiments, the method relates to reducing the patient's NT-proBNP byat least 70%. In some embodiments, the method relates to reducing thepatient's NT-proBNP by at least 75%. In some embodiments, the methodrelates to reducing the patient's NT-proBNP by at least 80%. In someembodiments, the method relates to reducing the patient's NT-proBNP byat least 85%. In some embodiments, the method relates to reducing thepatient's NT-proBNP by at least 90%. In some embodiments, the methodrelates to reducing the patient's NT-proBNP by at least 95%. In someembodiments, the method relates to reducing the patient's NT-proBNP byat least 100%. In some embodiments, the method relates to reducing thepatient's NT-proBNP levels to normal levels. In some embodiments, normallevels of NT-proBNP is <100 pg/ml. In some embodiments, the methodrelates to reducing the patient's NT-proBNP levels to less than 300ng/L.

Smooth Muscle Hypertrophy

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the patient has smoothmuscle hypertrophy. In some embodiments, the disclosure relates tomethods of adjusting one or more parameters in the PcPH patient toward amore normal level (e.g., normal as compared to healthy people of similarage and sex), comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1). In some embodiments, the methodrelates to decreasing smooth muscle hypertrophy in the patient. In someembodiments, the method relates to decreasing the patient's smoothmuscle hypertrophy by least 1% (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). In someembodiments, the method relates to decreasing the patient's smoothmuscle hypertrophy by at least 1%. In some embodiments, the methodrelates to decreasing the patient's smooth muscle hypertrophy by atleast 5%. In some embodiments, the method relates to decreasing thepatient's smooth muscle hypertrophy by at least 10%. In someembodiments, the method relates to decreasing the patient's smoothmuscle hypertrophy by at least 15%. In some embodiments, the methodrelates to decreasing the patient's smooth muscle hypertrophy by atleast 20%. In some embodiments, the method relates to decreasing thepatient's smooth muscle hypertrophy by at least 25%. In someembodiments, the method relates to decreasing the patient's smoothmuscle hypertrophy by at least 30%. In some embodiments, the methodrelates to decreasing the patient's smooth muscle hypertrophy by atleast 35%. In some embodiments, the method relates to decreasing thepatient's smooth muscle hypertrophy by at least 40%. In someembodiments, the method relates to decreasing the patient's smoothmuscle hypertrophy by at least 45%. In some embodiments, the methodrelates to decreasing the patient's smooth muscle hypertrophy by atleast 50%. In some embodiments, the method relates to decreasing thepatient's smooth muscle hypertrophy by at least 55%. In someembodiments, the method relates to decreasing the patient's smoothmuscle hypertrophy by at least 60%. In some embodiments, the methodrelates to decreasing the patient's smooth muscle hypertrophy by atleast 65%. In some embodiments, the method relates to decreasing thepatient's smooth muscle hypertrophy by at least 70%. In someembodiments, the method relates to decreasing the patient's smoothmuscle hypertrophy by at least 75%. In some embodiments, the methodrelates to decreasing the patient's smooth muscle hypertrophy by atleast 80%. In some embodiments, the method relates to decreasing thepatient's smooth muscle hypertrophy by at least 85%. In someembodiments, the method relates to decreasing the patient's smoothmuscle hypertrophy by at least 90%. In some embodiments, the methodrelates to decreasing the patient's smooth muscle hypertrophy by atleast 95%. In some embodiments, the method relates to decreasing thepatient's smooth muscle hypertrophy by at least 100%.

Pulmonary Arteriole Muscularity

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the patient has increasedpulmonary arteriole muscularity. In some embodiments, the disclosurerelates to methods of adjusting one or more parameters in the PcPHpatient toward a more normal level (e.g., normal as compared to healthypeople of similar age and sex), comprising administering to a patient inneed thereof an effective amount of an ActRII polypeptide (e.g., anamino acid sequence that is at least 90% identical to an amino acidsequence corresponding to residues 30-110 of SEQ ID NO: 1). In someembodiments, the method relates to decreasing pulmonary arteriolemuscularity in the patient. In some embodiments, the method relates todecreasing the patient's pulmonary arteriole muscularity by least 1%(e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or 100%). In some embodiments, the method relates todecreasing the patient's pulmonary arteriole muscularity by at least 1%.In some embodiments, the method relates to decreasing the patient'spulmonary arteriole muscularity by at least 5%. In some embodiments, themethod relates to decreasing the patient's pulmonary arteriolemuscularity by at least 10%. In some embodiments, the method relates todecreasing the patient's pulmonary arteriole muscularity by at least15%. In some embodiments, the method relates to decreasing the patient'spulmonary arteriole muscularity by at least 20%. In some embodiments,the method relates to decreasing the patient's pulmonary arteriolemuscularity by at least 25%. In some embodiments, the method relates todecreasing the patient's pulmonary arteriole muscularity by at least30%. In some embodiments, the method relates to decreasing the patient'spulmonary arteriole muscularity by at least 35%. In some embodiments,the method relates to decreasing the patient's pulmonary arteriolemuscularity by at least 40%. In some embodiments, the method relates todecreasing the patient's pulmonary arteriole muscularity by at least45%. In some embodiments, the method relates to decreasing the patient'spulmonary arteriole muscularity by at least 50%. In some embodiments,the method relates to decreasing the patient's pulmonary arteriolemuscularity by at least 55%. In some embodiments, the method relates todecreasing the patient's pulmonary arteriole muscularity by at least60%. In some embodiments, the method relates to decreasing the patient'spulmonary arteriole muscularity by at least 65%. In some embodiments,the method relates to decreasing the patient's pulmonary arteriolemuscularity by at least 70%. In some embodiments, the method relates todecreasing the patient's pulmonary arteriole muscularity by at least75%. In some embodiments, the method relates to decreasing the patient'spulmonary arteriole muscularity by at least 80%. In some embodiments,the method relates to decreasing the patient's pulmonary arteriolemuscularity by at least 85%. In some embodiments, the method relates todecreasing the patient's pulmonary arteriole muscularity by at least90%. In some embodiments, the method relates to decreasing the patient'spulmonary arteriole muscularity by at least 95%. In some embodiments,the method relates to decreasing the patient's pulmonary arteriolemuscularity by at least 100%.

Rate of Hospitalization

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the method reduces thepatient's hospitalization rate by at least 1% (e.g., 1%, 2%, 3%, 4%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100%. In some embodiments, the method relates toreducing the patient's hospitalization rate by at least 1%. In someembodiments, the method relates to reducing the patient'shospitalization rate by at least 2%. In some embodiments, the methodrelates to reducing the patient's hospitalization rate by at least 3%.In some embodiments, the method relates to reducing the patient'shospitalization rate by at least 4%. In some embodiments, the methodrelates to reducing the patient's hospitalization rate by at least 5%.In some embodiments, the method relates to reducing the patient'shospitalization rate by at least 10%. In some embodiments, the methodrelates to reducing the patient's hospitalization rate by at least 15%.In some embodiments, the method relates to reducing the patient'shospitalization rate by at least 20%. In some embodiments, the methodrelates to reducing the patient's hospitalization rate by at least 25%.In some embodiments, the method relates to reducing the patient'shospitalization rate by at least 30%. In some embodiments, the methodrelates to reducing the patient's hospitalization rate by at least 35%.In some embodiments, the method relates to reducing the patient'shospitalization rate by at least 40%. In some embodiments, the methodrelates to reducing the patient's hospitalization rate by at least 45%.In some embodiments, the method relates to reducing the patient'shospitalization rate by at least 50%. In some embodiments, the methodrelates to reducing the patient's hospitalization rate by at least 55%.In some embodiments, the method relates to reducing the patient'shospitalization rate by at least 60%. In some embodiments, the methodrelates to reducing the patient's hospitalization rate by at least 65%.In some embodiments, the method relates to reducing the patient'shospitalization rate by at least 70%. In some embodiments, the methodrelates to reducing the patient's hospitalization rate by at least 75%.In some embodiments, the method relates to reducing the patient'shospitalization rate by at least 80%. In some embodiments, the methodrelates to reducing the patient's hospitalization rate by at least 85%.In some embodiments, the method relates to reducing the patient'shospitalization rate by at least 90%. In some embodiments, the methodrelates to reducing the patient's hospitalization rate by at least 95%.In some embodiments, the method relates to reducing the patient'shospitalization rate by at least 100%. In some embodiments, the methodreduces the risk of hospitalization for one or more complicationsassociated with PcPH.

Quality of Life

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the method increases thepatient's quality of life by at least 1% (e.g., 1%, 2%, 3%, 4%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%). In some embodiments, the method relates toincreasing the patient's quality of life by at least 1%. In someembodiments, the method relates to increasing the patient's quality oflife by at least 2%. In some embodiments, the method relates toincreasing the patient's quality of life by at least 3%. In someembodiments, the method relates to increasing the patient's quality oflife by at least 4%. In some embodiments, the method relates toincreasing the patient's quality of life by at least 5%. In someembodiments, the method relates to increasing the patient's quality oflife by at least 10%. In some embodiments, the method relates toincreasing the patient's quality of life by at least 15%. In someembodiments, the method relates to increasing the patient's quality oflife by at least 20%. In some embodiments, the method relates toincreasing the patient's quality of life by at least 25%. In someembodiments, the method relates to increasing the patient's quality oflife by at least 30%. In some embodiments, the method relates toincreasing the patient's quality of life by at least 35%. In someembodiments, the method relates to increasing the patient's quality oflife by at least 40%. In some embodiments, the method relates toincreasing the patient's quality of life by at least 45%. In someembodiments, the method relates to increasing the patient's quality oflife by at least 50%. In some embodiments, the method relates toincreasing the patient's quality of life by at least 55%. In someembodiments, the method relates to increasing the patient's quality oflife by at least 60%. In some embodiments, the method relates toincreasing the patient's quality of life by at least 65%. In someembodiments, the method relates to increasing the patient's quality oflife by at least 70%. In some embodiments, the method relates toincreasing the patient's quality of life by at least 75%. In someembodiments, the method relates to increasing the patient's quality oflife by at least 80%. In some embodiments, the method relates toincreasing the patient's quality of life by at least 85%. In someembodiments, the method relates to increasing the patient's quality oflife by at least 90%. In some embodiments, the method relates toincreasing the patient's quality of life by at least 95%. In someembodiments, the method relates to increasing the patient's quality oflife by at least 100%.

In some embodiments, the patient's quality of life is measured using theCambridge Pulmonary Hypertension Outcome Review (CAMPHOR). In someembodiments, the patient's quality of life is measured usingPAH-SYMPACT®. In some embodiments, the patient's quality of life ismeasured using the Medical Outcomes Survey Short Form-36 (SF-36). Insome embodiments, the patient's quality of life is measured using theEuro Quality of Life (EuroQol). In some embodiments, the patient'squality of life is measured using the Euro Quality of Life—5 dimensions(EQ-5D). In some embodiments, the patient's quality of life is measuredusing the Euro Quality of Life—5 dimensions 5-levels (EQ-5D-5L). In someembodiments, the patient's quality of life is measured using the KansasCity Cardiomyopathy Questionnaire (KCCQ).

Diastolic Function

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the method increases thepatient's LV diastolic function by at least 5% (e.g., 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). Insome embodiments, the method relates to increasing the patient's LVdiastolic function by at least 5%. In some embodiments, the methodrelates to increasing the patient's LV diastolic function by at least10%. In some embodiments, the method relates to increasing the patient'sLV diastolic function by at least 15%. In some embodiments, the methodrelates to increasing the patient's LV diastolic function by at least20%. In some embodiments, the method relates to increasing the patient'sLV diastolic function by at least 25%. In some embodiments, the methodrelates to increasing the patient's LV diastolic function by at least30%. In some embodiments, the method relates to increasing the patient'sLV diastolic function by at least 35%. In some embodiments, the methodrelates to increasing the patient's LV diastolic function by at least40%. In some embodiments, the method relates to increasing the patient'sLV diastolic function by at least 45%. In some embodiments, the methodrelates to increasing the patient's LV diastolic function by at least50%. In some embodiments, the method relates to increasing the patient'sLV diastolic function by at least 55%. In some embodiments, the methodrelates to increasing the patient's LV diastolic function by at least60%. In some embodiments, the method relates to increasing the patient'sLV diastolic function by at least 65%. In some embodiments, the methodrelates to increasing the patient's LV diastolic function by at least70%. In some embodiments, the method relates to increasing the patient'sLV diastolic function by at least 75%. In some embodiments, the methodrelates to increasing the patient's LV diastolic function by at least80%. In some embodiments, the method relates to increasing the patient'sLV diastolic function by at least 85%. In some embodiments, the methodrelates to increasing the patient's LV diastolic function by at least90%. In some embodiments, the method relates to increasing the patient'sLV diastolic function by at least 95%. In some embodiments, the methodrelates to increasing the patient's LV diastolic function by at least100%.

Ejection Fraction

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the patient has an ejectionfraction of less than 10% (e.g., 10, 15, 20, 25, 30, 35, 40, or 45%). Insome embodiments, the method relates to patient's having an ejectionfraction of less than 10%. In some embodiments, the method relates topatient's having an ejection fraction of less than 15%. In someembodiments, the method relates to patient's having an ejection fractionof less than 20%. In some embodiments, the method relates to patient'shaving an ejection fraction of less than 25%. In some embodiments, themethod relates to patient's having an ejection fraction of less than30%. In some embodiments, the method relates to patient's having anejection fraction of less than 35%. In some embodiments, the methodrelates to patient's having an ejection fraction of less than 40%. Insome embodiments, the method relates to patient's having an ejectionfraction of less than 45%. In some embodiments, the method relates topatient's having an ejection fraction of less than 50%. In someembodiments, the method relates to patient's having an ejection fractionof less than 55%.

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the patient has an ejectionfraction of at least 35% (e.g., 35, 40, 45, 50, or 55%). In someembodiments, the method relates to patient's having an ejection fractionof at least 35%. In some embodiments, the method relates to patient'shaving an ejection fraction of at least 40%. In some embodiments, themethod relates to patient's having an ejection fraction of at least 45%.In some embodiments, the method relates to patient's having an ejectionfraction of at least 50%. In some embodiments, the method relates topatient's having an ejection fraction of at least 55%. In someembodiments, the ejection fraction is the right ventricular ejectionfraction. In some embodiments, the ejection fraction is the leftventricular ejection fraction (LVEF). In some embodiments, the ejectionfraction is measured using an echocardiogram. In some embodiments, thepatient has a preserved left ventricular ejection fraction.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., >50% ejection fraction), comprising administering to apatient in need thereof an effective amount of an ActRII polypeptide(e.g., an amino acid sequence that is at least 90% identical to an aminoacid sequence corresponding to residues 30-110 of SEQ ID NO: 1). In someembodiments, the method relates to increasing the patient's ejectionfraction by least 1%. In some embodiments, the method relates toincreasing the patient's ejection fraction by at least 5%. In someembodiments, the method relates to increasing the patient's ejectionfraction by at least 10%. In some embodiments, the method relates toincreasing the patient's ejection fraction by at least 15%. In someembodiments, the method relates to increasing the patient's ejectionfraction by at least 20%. In some embodiments, the method relates toincreasing the patient's ejection fraction by at least 25%. In someembodiments, the method relates to increasing the patient's ejectionfraction by at least 30%. In some embodiments, the method relates toincreasing the patient's ejection fraction by at least 35%. In someembodiments, the method relates to increasing the patient's ejectionfraction by at least 40%. In some embodiments, the method relates toincreasing the patient's ejection fraction by at least 45%. In someembodiments, the method relates to increasing the patient's ejectionfraction by at least 50%. In some embodiments, the method relates toincreasing the patient's ejection fraction by at least 55%. In someembodiments, the method relates to increasing the patient's ejectionfraction by at least 60%. In some embodiments, the method relates toincreasing the patient's ejection fraction by at least 65%. In someembodiments, the method relates to increasing the patient's ejectionfraction by at least 70%. In some embodiments, the method relates toincreasing the patient's ejection fraction by at least 75%. In someembodiments, the method relates to increasing the patient's ejectionfraction by at least 80%. In some embodiments, the method relates toincreasing the patient's ejection fraction by at least 85%. In someembodiments, the method relates to increasing the patient's ejectionfraction by at least 90%. In some embodiments, the method relates toincreasing the patient's ejection fraction by at least 95%. In someembodiments, the method relates to increasing the patient's ejectionfraction by at least 100%.

Ventricular Function

In certain aspects, the disclosure relates to methods of improving ormaintaining ventricular function (e.g., left ventricular function orright ventricular function) in PcPH comprising administering to apatient in need thereof an effective amount of an ActRII polypeptide(e.g., an amino acid sequence that is at least 90% identical to an aminoacid sequence corresponding to residues 30-110 of SEQ ID NO: 1).Echocardiography is a useful noninvasive screening tool for determiningthe severity of pulmonary hypertension in a patient. Improvement ormaintenance of ventricular function (e.g., left ventricular function orright ventricular function) can be assessed by many echocardiographicmeasurements. One such quantitative approach to assess ventricularfunction is the measurement of the tricuspid annular plane systolicexcursion (TAPSE). The TAPSE estimates RV systolic function by measuringthe level of systolic excursion of the lateral tricuspid valve annulustowards the apex. Other echocardiographic measurements that may be usedto assess maintenance and/or improvements in ventricular functioninclude, but are not limited to, right ventricular fractional areachange (RVFAC), right ventricular end-diastolic area (RVEDA), rightventricular end-systolic area (RVESA), right ventricular free wallthickness (RVFWT), right ventricular ejection fraction (RVEF), rightventricular-pulmonary artery (RV-PA) coupling, pulmonary arterialsystolic pressure (PASP), right ventricular systolic pressure (RVSP),pulmonary artery acceleration time (PAAT), tricuspid regurgitationvelocity (TRV), left ventricular hypertrophy, and right ventricularhypertrophy.

TAPSE

The tricuspid annular plane systolic excursion (TAPSE) can be obtainedusing echocardiography and represents a measure of RV longitudinalfunction. The TAPSE has previously been shown to have good correlationswith parameters estimating RV global systolic function. A TAPSE <17 mmis highly suggestive of RV systolic dysfunction. In some embodiments, animprovement or maintenance of right ventricular function in a PcPHpatient is measured as an increase in TAPSE. In some embodiments, a PcPHpatient with an improvement or maintenance of right ventricular functionhas a TAPSE between 20 mm-28 mm. In some embodiments, a PcPH patientwith an improvement or maintenance of right ventricular function has aTAPSE of at least 20 mm. In some embodiments, a PcPH patient with animprovement or maintenance of right ventricular function has a TAPSE ofat least 22 mm. In some embodiments, a PcPH patient with an improvementor maintenance of right ventricular function has a TAPSE of at least 24mm. In some embodiments, a PcPH patient with an improvement ormaintenance of right ventricular function has a TAPSE of at least 26 mm.In some embodiments, a PcPH patient with an improvement or maintenanceof right ventricular function has a TAPSE of at least 28 mm. In someembodiments, the TAPSE is measured using echocardiography.

In some embodiments, a PcPH patient with an improvement or maintenanceof right ventricular function has a TAPSE between 16 mm-30 mm. In someembodiments, a PcPH patient with an improvement or maintenance of rightventricular function has a TAPSE between 18 mm-28 mm. In someembodiments, a PcPH patient with an improvement or maintenance of rightventricular function has a TAPSE of at least 18 mm. In some embodiments,the TAPSE is measured using echocardiography.

PASP and RVSP

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPHcomprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1), wherein the patient has a pulmonary arterialsystolic pressure (PASP) of at least 30 mmHg (e.g., 30, 35, 40, 45, 50,55, 60, 65, 70, 75, or 80 mmHg). In some embodiments, the method relatesto patients having a PASP of at least 30 mmHg. In some embodiments, themethod relates to patients having a PASP of at least 35 mmHg. In someembodiments, the method relates to patients having a PASP of at least 40mmHg. In some embodiments, the method relates to patients having a PASPof at least 45 mmHg. In some embodiments, the method relates to patientshaving a PASP of at least 50 mmHg. In some embodiments, the methodrelates to patients having a PASP of at least 55 mmHg. In someembodiments, the method relates to patients having a PASP of at least 60mmHg. In some embodiments, the method relates to patients having a PASPof at least 65 mmHg. In some embodiments, the method relates to patientshaving a PASP of at least 70 mmHg. In some embodiments, the methodrelates to patients having a PASP of at least 75 mmHg. In someembodiments, the method relates to patients having a PASP of at least 80mmHg. In some embodiments, the PASP is a resting PASP. In someembodiments, the PASP is determined using the tricuspid regurgitationvelocity (TRV) and right arterial (RA) pressure. In some embodiments,the PASP is determined using the following formula:

PASP=TRV²×4+RA pressure

TRV has been shown to correlate with PASP at rest and with exercise. Thepressure gradient between the right ventricle and the right atrium canbe calculated using the modified Bernoulli equation (Δp=4V²).

In some embodiments, the right ventricular systolic pressure (RVSP) isequal to PASP. In some embodiments, the RVSP is measured in the absenceof right ventricular outflow tract obstruction. In some embodiments, theRVSP is determined using the following formula:

RVSP=4V ²+RAP

In the above formula, V represents the peak tricuspid regurgitant jetvelocity and RAP is the mean right atrial pressure. RVSP is frequentlyused for estimating PASP.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates toimproving the pulmonary arterial systolic pressure (PASP) in thepatient. In some embodiments, the method relates to reducing PASP. Insome embodiments, the method relates to reducing the patient's PASP byat least 1 mmHg (e.g., 1, 2, 3, 5, 7, 10, 12, 15, 20, 25, 30, or 35mmHg). In some embodiments, the method relates to reducing the patient'sPASP by at least 2 mmHg. In some embodiments, the method relates toreducing the patient's PASP by at least 3 mmHg. In certain embodiments,the method relates to reducing the patient's PASP by at least 5 mmHg. Incertain embodiments, the method relates to reducing the patient's PASPby at least 7 mmHg. In certain embodiments, the method relates toreducing the patient's PASP by at least 10 mmHg. In certain embodiments,the method relates to reducing the patient's PASP by at least 12 mmHg.In certain embodiments, the method relates to reducing the patient'sPASP by at least 15 mmHg. In certain embodiments, the method relates toreducing the patient's PASP by at least 20 mmHg. In certain embodiments,the method relates to reducing the patient's PASP by at least 25 mmHg.In certain embodiments, the method relates to reducing the patient'sPASP by at least 30 mmHg. In certain embodiments, the method relates toreducing the patient's PASP by at least 35 mmHg.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates toreducing the patient's PASP by least 1% (e.g., 1, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). In someembodiments, the method relates to reducing the patient's PASP by atleast 1%. In some embodiments, the method relates to reducing thepatient's PASP by at least 5%. In some embodiments, the method relatesto reducing the patient's PASP by at least 10%. In some embodiments, themethod relates to reducing the patient's PASP by at least 15%. In someembodiments, the method relates to reducing the patient's PASP by atleast 20%. In some embodiments, the method relates to reducing thepatient's PASP by at least 25%. In some embodiments, the method relatesto reducing the patient's PASP by at least 30%. In some embodiments, themethod relates to reducing the patient's PASP by at least 35%. In someembodiments, the method relates to reducing the patient's PASP by atleast 40%. In some embodiments, the method relates to reducing thepatient's PASP by at least 45%. In some embodiments, the method relatesto reducing the patient's PASP by at least 50%. In some embodiments, themethod relates to reducing the patient's PASP by at least 55%. In someembodiments, the method relates to reducing the patient's PASP by atleast 60%. In some embodiments, the method relates to reducing thepatient's PASP by at least 65%. In some embodiments, the method relatesto reducing the patient's PASP by at least 70%. In some embodiments, themethod relates to reducing the patient's PASP by at least 75%. In someembodiments, the method relates to reducing the patient's PASP by atleast 80%. In some embodiments, the method relates to reducing thepatient's PASP by at least 85%. In some embodiments, the method relatesto reducing the patient's PASP by at least 90%. In some embodiments, themethod relates to reducing the patient's PASP by at least 95%. In someembodiments, the method relates to reducing the patient's PASP by atleast 100%.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates toimproving the right ventricular systolic pressure (RVSP) in the patient.In some embodiments, the method relates to reducing RVSP. In someembodiments, the method relates to reducing the patient's RVSP by atleast 1 mmHg (e.g., 1, 2, 3, 5, 7, 10, 12, 15, 20, 25, 30, or 35 mmHg).In some embodiments, the method relates to reducing the patient's RVSPby at least 2 mmHg. In some embodiments, the method relates to reducingthe patient's RVSP by at least 3 mmHg. In certain embodiments, themethod relates to reducing the patient's RVSP by at least 5 mmHg. Incertain embodiments, the method relates to reducing the patient's RVSPby at least 7 mmHg. In certain embodiments, the method relates toreducing the patient's RVSP by at least 10 mmHg. In certain embodiments,the method relates to reducing the patient's RVSP by at least 12 mmHg.In certain embodiments, the method relates to reducing the patient'sRVSP by at least 15 mmHg. In certain embodiments, the method relates toreducing the patient's RVSP by at least 20 mmHg. In certain embodiments,the method relates to reducing the patient's RVSP by at least 25 mmHg.In certain embodiments, the method relates to reducing the patient'sRVSP by at least 30 mmHg. In certain embodiments, the method relates toreducing the patient's RVSP by at least 35 mmHg.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates toreducing the patient's RVSP by least 1% (e.g., 1, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). In someembodiments, the method relates to reducing the patient's RVSP by atleast 5%. In some embodiments, the method relates to reducing thepatient's RVSP by at least 10%. In some embodiments, the method relatesto reducing the patient's RVSP by at least 15%. In some embodiments, themethod relates to reducing the patient's RVSP by at least 20%. In someembodiments, the method relates to reducing the patient's RVSP by atleast 25%. In some embodiments, the method relates to reducing thepatient's RVSP by at least 30%. In some embodiments, the method relatesto reducing the patient's RVSP by at least 35%. In some embodiments, themethod relates to reducing the patient's RVSP by at least 40%. In someembodiments, the method relates to reducing the patient's RVSP by atleast 45%. In some embodiments, the method relates to reducing thepatient's RVSP by at least 50%. In some embodiments, the method relatesto reducing the patient's RVSP by at least 55%. In some embodiments, themethod relates to reducing the patient's RVSP by at least 60%. In someembodiments, the method relates to reducing the patient's RVSP by atleast 65%. In some embodiments, the method relates to reducing thepatient's RVSP by at least 70%. In some embodiments, the method relatesto reducing the patient's RVSP by at least 75%. In some embodiments, themethod relates to reducing the patient's RVSP by at least 80%. In someembodiments, the method relates to reducing the patient's RVSP by atleast 85%. In some embodiments, the method relates to reducing thepatient's RVSP by at least 90%. In some embodiments, the method relatesto reducing the patient's RVSP by at least 95%. In some embodiments, themethod relates to reducing the patient's RVSP by at least 100%.

RV-PA Coupling

Right ventricular dysfunction can occur in PcPH and is a factoraffecting prognosis. Energy transfer between ventricle contractility andarterial afterload is termed coupling. Energy transfer specificallybetween the right ventricle (RV) and pulmonary artery is termed rightventricle-pulmonary artery (RV-PA) coupling. In some embodiments, rightventricular dysfunction is due to a decrease in RV-PA coupling. RV-PAcoupling can be estimated non-invasively as a ratio of TAPSE/PASPvalues. In some embodiments, a TAPSE/PASP ratio of ≥0.31 mm/mm Hg may beassociated with a better prognosis and reduced risk of clinicalworsening. In some embodiments, the improvement in RV-PA coupling is dueto an improvement in PASP. In some embodiments, the calculation of RV-PAcoupling is dependent upon paired results for three parameters (e.g.,TRV, RAP, and TAPSE).

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPHcomprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1), wherein the patient has a TAPSE/PASP ratio lessthan 0.31 mm/mmHg (e.g., 0.3, 0.25, 0.2, 0.15, or 0.1 mm/mmHg). In someembodiments, the method relates to patients having a TAPSE/PASP ratioless than 0.31 mm/mmHg. In some embodiments, the method relates topatients having a TAPSE/PASP ratio less than 0.3 mm/mmHg. In someembodiments, the method relates to patients having a TAPSE/PASP ratioless than 0.25 mm/mmHg. In some embodiments, the method relates topatients having a TAPSE/PASP ratio less than 0.2 mm/mmHg. In someembodiments, the method relates to patients having a TAPSE/PASP ratioless than 0.15 mm/mmHg. In some embodiments, the method relates topatients having a TAPSE/PASP ratio less than 0.1 mm/mmHg. In someembodiments, the method relates to patients having a decreasedTAPSE/PASP ratio as compared to a normal TAPSE/PASP ratio.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates toimproving or maintaining the right ventricular function in the patient.In some embodiments, a PcPH patient with an improvement or maintenanceof right ventricular function has a TAPSE/PASP ratio greater than 0.3mm/mmHg (e.g., greater than 0.31, 0.32, 0.33, 0.34, or 0.35 mm/mmHg). Insome embodiments, a PcPH patient with an improvement or maintenance ofright ventricular function has a TAPSE/PASP ratio greater than 0.31mm/mmHg. In some embodiments, a PcPH patient with an improvement ormaintenance of right ventricular function has a TAPSE/PASP ratio greaterthan 0.32 mm/mmHg. In some embodiments, a PcPH patient with animprovement or maintenance of right ventricular function has aTAPSE/PASP ratio greater than 0.33 mm/mmHg. In some embodiments, a PcPHpatient with an improvement or maintenance of right ventricular functionhas a TAPSE/PASP ratio greater than 0.34 mm/mmHg. In some embodiments, aPcPH patient with an improvement or maintenance of right ventricularfunction has a TAPSE/PASP ratio greater than 0.35 mm/mmHg. In someembodiments, the improvement in right ventricular function is anincrease in TAPSE/PASP ratio. In some embodiments, the method relates toincreasing the TAPSE/PASP ratio. In some embodiments, the method relatesto increasing the patient's TAPSE/PASP ratio by at least 0.05 mm/mmHg.In some embodiments, the method relates to increasing the patient'sTAPSE/PASP ratio by at least 0.07 mm/mmHg. In some embodiments, themethod relates to increasing the patient's TAPSE/PASP ratio by at least0.10 mm/mmHg. In some embodiments, the method relates to increasing thepatient's TAPSE/PASP ratio by at least 0.12 mm/mmHg. In someembodiments, the method relates to increasing the patient's TAPSE/PASPratio by at least 0.15 mm/mmHg. In some embodiments, the method relatesto increasing the patient's TAPSE/PASP ratio by at least 0.18 mm/mmHg.In some embodiments, the method relates to increasing the patient'sTAPSE/PASP ratio by at least 0.20 mm/mmHg.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates toincreasing the patient's TAPSE/PASP ratio by least 1% (e.g., 1, 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or100%). In some embodiments, the method relates to increasing thepatient's TAPSE/PASP ratio by at least 5%. In some embodiments, themethod relates to increasing the patient's TAPSE/PASP ratio by at least10%. In some embodiments, the method relates to increasing the patient'sTAPSE/PASP ratio by at least 15%. In some embodiments, the methodrelates to increasing the patient's TAPSE/PASP ratio by at least 20%. Insome embodiments, the method relates to increasing the patient'sTAPSE/PASP ratio by at least 25%. In some embodiments, the methodrelates to increasing the patient's TAPSE/PASP ratio by at least 30%. Insome embodiments, the method relates to increasing the patient'sTAPSE/PASP ratio by at least 35%. In some embodiments, the methodrelates to increasing the patient's TAPSE/PASP ratio by at least 40%. Insome embodiments, the method relates to increasing the patient'sTAPSE/PASP ratio by at least 45%. In some embodiments, the methodrelates to increasing the patient's TAPSE/PASP ratio by at least 50%. Insome embodiments, the method relates to increasing the patient'sTAPSE/PASP ratio by at least 55%. In some embodiments, the methodrelates to increasing the patient's TAPSE/PASP ratio by at least 60%. Insome embodiments, the method relates to increasing the patient'sTAPSE/PASP ratio by at least 65%. In some embodiments, the methodrelates to increasing the patient's TAPSE/PASP ratio by at least 70%. Insome embodiments, the method relates to increasing the patient'sTAPSE/PASP ratio by at least 75%. In some embodiments, the methodrelates to increasing the patient's TAPSE/PASP ratio by at least 80%. Insome embodiments, the method relates to increasing the patient'sTAPSE/PASP ratio by at least 85%. In some embodiments, the methodrelates to increasing the patient's TAPSE/PASP ratio by at least 90%. Insome embodiments, the method relates to increasing the patient'sTAPSE/PASP ratio by at least 100%.

RVFAC, RVEDA, and RVESA

Right ventricular fractional area change (RVFAC) is a non-invasivequantitative measure of right ventricular function. RVFAC can becalculated using the formula [(RVEDA-RVESA)/RVEDA]*100. In someembodiments, the RVFAC is measured using echocardiography. In someembodiments, normal RVFAC is approximately 47.5±8.6% in men andapproximately 50.9±8.0% in women. See, e.g., Kou S, et al. EuropeanHeart Journal—Cardiovascular Imaging. 2014 Jun. 1; 15(6):680-90. In someembodiments, PcPH patients have a decrease in RVFAC.

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the patient has a RVFAC ofless than 20% (e.g., 20, 25, 30, 35, or 40%). In some embodiments, themethod relates to patients having a RVFAC of less than 25%. In someembodiments, the method relates to patients having a RVFAC of less than30%. In some embodiments, the method relates to patients having a RVFACof less than 35%. In some embodiments, the method relates to patientshaving a RVFAC of less than 40%.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates toimproving or maintaining the right ventricular function in the patient.In some embodiments, the improvement or maintenance of right ventricularfunction is due to an increase in right ventricular fractional areachange (RVFAC). In some embodiments, a PcPH patient with an improvementor maintenance of right ventricular function has a RVFAC between 32-56%.In some embodiments, a PcPH patient with an improvement or maintenanceof right ventricular function has a RVFAC of at least 32%. In someembodiments, a PcPH patient with an improvement or maintenance of rightventricular function has a RVFAC of at least 34%. In some embodiments, aPcPH patient with an improvement or maintenance of right ventricularfunction has a RVFAC of at least 35%. In some embodiments, a PcPHpatient with an improvement or maintenance of right ventricular functionhas a RVFAC of at least 36%. In some embodiments, a PcPH patient with animprovement or maintenance of right ventricular function has a RVFAC ofat least 38%. In some embodiments, a PcPH patient with an improvement ormaintenance of right ventricular function has a RVFAC of at least 40%.In some embodiments, a PcPH patient with an improvement or maintenanceof right ventricular function has a RVFAC of at least 42%. In someembodiments, a PcPH patient with an improvement or maintenance of rightventricular function has a RVFAC of at least 44%. In some embodiments, aPcPH patient with an improvement or maintenance of right ventricularfunction has a RVFAC of at least 46%. In some embodiments, a PcPHpatient with an improvement or maintenance of right ventricular functionhas a RVFAC of at least 48%. In some embodiments, a PcPH patient with animprovement or maintenance of right ventricular function has a RVFAC ofat least 50%. In some embodiments, a PcPH patient with an improvement ormaintenance of right ventricular function has a RVFAC of at least 52%.In some embodiments, a PcPH patient with an improvement or maintenanceof right ventricular function has a RVFAC of at least 54%. In someembodiments, a PcPH patient with an improvement or maintenance of rightventricular function has a RVFAC of at least 56%.

In some embodiments, the disclosure relates to methods of adjusting oneor more echocardiogram parameters in the PcPH patient toward a morenormal level (e.g., normal as compared to healthy people of similar ageand sex), comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1). In some embodiments, the methodrelates to decreasing the patient's RVEDA by least 1% (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20%). In some embodiments, themethod relates to increasing the patient's RVFAC by least 2%. In someembodiments, the method relates to increasing the patient's RVFAC byleast 3%. In some embodiments, the method relates to increasing thepatient's RVFAC by least 4%. In some embodiments, the method relates toincreasing the patient's RVFAC by least 5%. In some embodiments, themethod relates to increasing the patient's RVFAC by least 6%. In someembodiments, the method relates to increasing the patient's RVFAC byleast 7%. In some embodiments, the method relates to increasing thepatient's RVFAC by least 8%. In some embodiments, the method relates toincreasing the patient's RVFAC by least 9%. In some embodiments, themethod relates to increasing the patient's RVFAC by least 10%. In someembodiments, the method relates to increasing the patient's RVFAC byleast 12%. In some embodiments, the method relates to increasing thepatient's RVFAC by least 14%. In some embodiments, the method relates toincreasing the patient's RVFAC by least 16%. In some embodiments, themethod relates to increasing the patient's RVFAC by least 18%. In someembodiments, the method relates to increasing the patient's RVFAC byleast 20%.

In some embodiments, the improvement in right ventricular function isdue to an increase in ejection fraction. In some embodiments, theimprovement in right ventricular function is due to an increase inejection fraction and an increase in the patient's RVFAC.

The right ventricular end-diastolic area (RVEDA) can be measured usingechocardiography. In some embodiments, normal RVEDA is approximately18.2±4.3 cm² in men and approximately 14.8±3.5 cm² in women. See, e.g.,Kou S, et al. European Heart Journal—Cardiovascular Imaging. 2014 Jun.1; 15(6):680-90.

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPHcomprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1), wherein the patient has a RVEDA of at least 22cm² (e.g., 22, 24, 26, 28, 30, 32, or 34 cm²). In some embodiments, themethod relates to patients having a RVEDA of at least 24 cm². In someembodiments, the method relates to patients having a RVEDA of at least26 cm². In some embodiments, the method relates to patients having aRVEDA of at least 28 cm². In some embodiments, the method relates topatients having a RVEDA of at least 30 cm². In some embodiments, themethod relates to patients having a RVEDA of at least 32 cm². In someembodiments, the method relates to patients having a RVEDA of at least34 cm². In some embodiments, the method relates to patients havingincreased RVEDA as compared to normal RVEDA.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates toimproving or maintaining the right ventricular function in the patient.In some embodiments, a patient with an improvement or maintenance ofright ventricular function has a RVEDA of 14-22 cm². In someembodiments, the improvement in right ventricular function is areduction in RVEDA. In some embodiments, the method relates to reducingthe RVEDA. In some embodiments, the method relates to reducing thepatients RVEDA by at least 1 cm². In some embodiments, the methodrelates to reducing the patients RVEDA by at least 2 cm². In someembodiments, the method relates to reducing the patients RVEDA by atleast 3 cm². In some embodiments, the method relates to reducing thepatients RVEDA by at least 4 cm². In some embodiments, the methodrelates to reducing the patients RVEDA by at least 5 cm². In someembodiments, the method relates to reducing the patients RVEDA by atleast 6 cm². In some embodiments, the method relates to reducing thepatients RVEDA by at least 7 cm². In some embodiments, the methodrelates to reducing the patients RVEDA by at least 8 cm². In someembodiments, the method relates to reducing the patients RVEDA by atleast 9 cm². In some embodiments, the method relates to reducing thepatients RVEDA by at least 10 cm².

In some embodiments, the disclosure relates to methods of adjusting oneor more echocardiogram parameters in the PcPH patient toward a morenormal level (e.g., normal as compared to healthy people of similar ageand sex), comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1). In some embodiments, the methodrelates to decreasing the patient's RVEDA by least 1% (e.g., 1, 5, 10,15, 20, 25, 30, 35, or 40%). In some embodiments, the method relates todecreasing the patient's RVEDA by at least 5%. In some embodiments, themethod relates to decreasing the patient's RVEDA by at least 10%. Insome embodiments, the method relates to decreasing the patient's RVEDAby at least 15%. In some embodiments, the method relates to decreasingthe patient's RVEDA by at least 20%. In some embodiments, the methodrelates to decreasing the patient's RVEDA by at least 25%. In someembodiments, the method relates to decreasing the patient's RVEDA by atleast 30%. In some embodiments, the method relates to decreasing thepatient's RVEDA by at least 35%. In some embodiments, the method relatesto decreasing the patient's RVEDA by at least 40%.

The right ventricular end-systolic area (RVESA) can be measured usingechocardiography. In some embodiments, normal RVESA is approximately9.6±2.8 cm² in men and approximately 7.3±2.3 cm² in women. See, e.g.,Kou S, et al. European Heart Journal—Cardiovascular Imaging. 2014 Jun.1; 15(6):680-90.

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPHcomprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1), wherein the patient has a RVESA of at least 12cm² (e.g., 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 cm²). In someembodiments, the method relates to patients having a RVESA of at least14 cm². In some embodiments, the method relates to patients having aRVESA of at least 16 cm². In some embodiments, the method relates topatients having a RVESA of at least 18 cm². In some embodiments, themethod relates to patients having a RVESA of at least 20 cm². In someembodiments, the method relates to patients having a RVESA of at least22 cm². In some embodiments, the method relates to patients having aRVESA of at least 24 cm². In some embodiments, the method relates topatients having a RVESA of at least 26 cm². In some embodiments, themethod relates to patients having a RVESA of at least 28 cm². In someembodiments, the method relates to patients having a RVESA of at least30 cm². In some embodiments, the method relates to patients having aRVESA of at least 32 cm². In some embodiments, the method relates topatients having increased RVESA as compared to normal RVESA.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates toimproving or maintaining the right ventricular function in the patient.In some embodiments, a patient with an improvement or maintenance ofright ventricular function has a RVESA of 7-20 cm². In some embodiments,the improvement in right ventricular function is a reduction in RVESA.In some embodiments, the method relates to reducing the RVESA. In someembodiments, the method relates to reducing the patient's RVESA by atleast 1 cm². In some embodiments, the method relates to reducing thepatient's RVESA by at least 2 cm². In some embodiments, the methodrelates to reducing the patient's RVESA by at least 3 cm². In someembodiments, the method relates to reducing the patient's RVESA by atleast 4 cm². In some embodiments, the method relates to reducing thepatient's RVESA by at least 5 cm². In some embodiments, the methodrelates to reducing the patient's RVESA by at least 6 cm². In someembodiments, the method relates to reducing the patient's RVESA by atleast 7 cm². In some embodiments, the method relates to reducing thepatient's RVESA by at least 8 cm². In some embodiments, the methodrelates to reducing the patient's RVESA by at least 9 cm². In someembodiments, the method relates to reducing the patient's RVESA by atleast 10 cm².

In some embodiments, the disclosure relates to methods of adjusting oneor more echocardiogram parameters in the PcPH patient toward a morenormal level (e.g., normal as compared to healthy people of similar ageand sex), comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1). In some embodiments, the methodrelates to decreasing the patient's RVESA by least 1% (e.g., 1, 2, 3, 4,5, 10, 15, 20, 25, 30, 35, or 40%). In some embodiments, the methodrelates to decreasing the patient's RVESA by at least 2%. In someembodiments, the method relates to decreasing the patient's RVESA by atleast 3%. In some embodiments, the method relates to decreasing thepatient's RVESA by at least 4%. In some embodiments, the method relatesto decreasing the patient's RVESA by at least 5%. In some embodiments,the method relates to decreasing the patient's RVESA by at least 10%. Insome embodiments, the method relates to decreasing the patient's RVESAby at least 15%. In some embodiments, the method relates to decreasingthe patient's RVESA by at least 20%. In some embodiments, the methodrelates to decreasing the patient's RVESA by at least 25%. In someembodiments, the method relates to decreasing the patient's RVESA by atleast 30%. In some embodiments, the method relates to decreasing thepatient's RVESA by at least 35%. In some embodiments, the method relatesto decreasing the patient's RVESA by at least 40%.

RVFWT

In patients with pulmonary hypertension, the right ventricle dilates inresponse to increased PAP and right ventricular remodeling. As thedisease progresses right ventricular hypertrophy develops, resulting inincreased right ventricle free wall thickness. In some embodiments, theright ventricular free wall thickness (RVFWT) can be measured usingechocardiography. In some embodiments, normal RVFWT is approximately0.22-0.42 cm in women and approximately 0.24-0.42 cm in men. See, e.g.,Lang R M, J Am Soc Echocardiogr. 2015; 28(1):1-39.e14.

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the patient has a RVFWT ofat least 0.42 cm (e.g., 0.42, 0.44, 0.46, 0.48, 0.50, 0.52, 0.54, 0.56,0.58, or 0.60 cm). In some embodiments, the method relates to patientshaving a RVFWT of at least 0.44 cm. In some embodiments, the methodrelates to patients having a RVFWT of at least 0.46 cm. In someembodiments, the method relates to patients having a RVFWT of at least0.48 cm. In some embodiments, the method relates to patients having aRVFWT of at least 0.50 cm. In some embodiments, the method relates topatients having a RVFWT of at least 0.52 cm. In some embodiments, themethod relates to patients having a RVFWT of at least 0.54 cm. In someembodiments, the method relates to patients having a RVFWT of at least0.56 cm. In some embodiments, the method relates to patients having aRVFWT of at least 0.58 cm. In some embodiments, the method relates topatients having a RVFWT of at least 0.60 cm. In some embodiments, themethod relates to patients having increased RVFWT as compared to normalRVFWT.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PcPH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates toimproving or maintaining the right ventricular function in the patient.In some embodiments, a patient with an improvement or maintenance ofright ventricular function has a RVFWT of between 0.22-0.42 cm. In someembodiments, the improvement in right ventricular function is areduction in RVFWT. In some embodiments, the method relates to reducingthe RVFWT. In some embodiments, the method relates to reducing thepatients RVFWT by at least 0.05 cm (e.g., 0.05, 0.1, 0.15, 0.2, 0.25,0.3, 0.35, or 0.4 cm). In some embodiments, the method relates toreducing the patients RVFWT by at least 0.1 cm. In some embodiments, themethod relates to reducing the patients RVFWT by at least 0.15 cm. Insome embodiments, the method relates to reducing the patients RVFWT byat least 0.2 cm. In some embodiments, the method relates to reducing thepatients RVFWT by at least 0.25 cm. In some embodiments, the methodrelates to reducing the patients RVFWT by at least 0.3 cm. In someembodiments, the method relates to reducing the patients RVFWT by atleast 0.35 cm. In some embodiments, the method relates to reducing thepatients RVFWT by at least 0.4 cm.

In some embodiments, the disclosure relates to methods of adjusting theRVFWT in the PcPH patient toward a more normal level (e.g., normal ascompared to healthy people of similar age and sex), comprisingadministering to a patient in need thereof an effective amount of anActRII polypeptide (e.g., an amino acid sequence that is at least 90%identical to an amino acid sequence corresponding to residues 30-110 ofSEQ ID NO: 1). In some embodiments, the method relates to decreasing thepatient's RVFWT by least 1% (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, or 75%). In some embodiments, the method relates todecreasing the patient's RVFWT by at least 5%. In some embodiments, themethod relates to decreasing the patient's RVFWT by at least 10%. Insome embodiments, the method relates to decreasing the patient's RVFWTby at least 15%. In some embodiments, the method relates to decreasingthe patient's RVFWT by at least 20%. In some embodiments, the methodrelates to decreasing the patient's RVFWT by at least 25%. In someembodiments, the method relates to decreasing the patient's RVFWT by atleast 30%. In some embodiments, the method relates to decreasing thepatient's RVFWT by at least 35%. In some embodiments, the method relatesto decreasing the patient's RVFWT by at least 40%. In some embodiments,the method relates to decreasing the patient's RVFWT by at least 45%. Insome embodiments, the method relates to decreasing the patient's RVFWTby at least 50%. In some embodiments, the method relates to decreasingthe patient's RVFWT by at least 55%. In some embodiments, the methodrelates to decreasing the patient's RVFWT by at least 60%. In someembodiments, the method relates to decreasing the patient's RVFWT by atleast 65%. In some embodiments, the method relates to decreasing thepatient's RVFWT by at least 70%. In some embodiments, the method relatesto decreasing the patient's RVFWT by at least 75%.

RVEF

Right ventricular ejection fraction is a global measure of RV systolicperformance. RVEF can be calculated using the RV end-diastolic volume(RVEDV) and RV end systolic volume (RVESV). Specifically, RVEF can becalculated using the following formula: RVEF(%)=((RVEDV−RVESV)/RVEDV)*100. Normal RVEF is approximately 56-65% inmen and 60-71% in women. See, e.g., Lang R M, J Am Soc Echocardiogr.2015; 28(1):1-39.e14. In some embodiments, the RVEF is measured usingechocardiography. In some embodiments, the disclosure relates to methodsof adjusting one or more hemodynamic parameters in the PcPH patienttoward a more normal level (e.g., normal as compared to healthy peopleof similar age and sex), comprising administering to a patient in needthereof an effective amount of an ActRII polypeptide (e.g., an aminoacid sequence that is at least 90% identical to an amino acid sequencecorresponding to residues 30-110 of SEQ ID NO: 1). In some embodiments,the method relates to improving or maintaining the right ventricularfunction in the patient. In some embodiments, a patient with animprovement or maintenance of right ventricular function has a RVEF of45-71%. In some embodiments, a patient with an improvement ormaintenance of right ventricular function has a RVEF of at least 45%. Insome embodiments, a patient with an improvement or maintenance of rightventricular function has a RVEF of at least 50%. In some embodiments, apatient with an improvement or maintenance of right ventricular functionhas a RVEF of at least 55%. In some embodiments, a patient with animprovement or maintenance of right ventricular function has a RVEF ofat least 60%. In some embodiments, a patient with an improvement ormaintenance of right ventricular function has a RVEF of at least 65%. Insome embodiments, a patient with an improvement or maintenance of rightventricular function has a RVEF of at least 70%.

In some embodiments, the disclosure relates to methods of adjusting oneor more echocardiogram parameters in the PcPH patient toward a morenormal level (e.g., normal as compared to healthy people of similar ageand sex), comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1). In some embodiments, the methodrelates to increasing the patient's RVEF by least 2%. In someembodiments, the method relates to increasing the patient's RVEF byleast 3%. In some embodiments, the method relates to increasing thepatient's RVEF by least 4%. In some embodiments, the method relates toincreasing the patient's RVEF by least 5%. In some embodiments, themethod relates to increasing the patient's RVEF by least 6%. In someembodiments, the method relates to increasing the patient's RVEF byleast 7%. In some embodiments, the method relates to increasing thepatient's RVEF by least 8%. In some embodiments, the method relates toincreasing the patient's RVEF by least 9%. In some embodiments, themethod relates to increasing the patient's RVEF by least 10%. In someembodiments, the method relates to increasing the patient's RVEF byleast 11%. In some embodiments, the method relates to increasing thepatient's RVEF by least 12%. In some embodiments, the method relates toincreasing the patient's RVEF by least 13%. In some embodiments, themethod relates to increasing the patient's RVEF by least 14%. In someembodiments, the method relates to increasing the patient's RVEF byleast 15%.

Right Ventricular Hypertrophy

In certain aspects, the improvement in right ventricular function ismeasured as a decrease in right ventricular hypertrophy. In someembodiment, the right ventricular hypertrophy is measured using theFulton Index (RV/(LV+S)).

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the patient has rightventricular hypertrophy. In some embodiments, the disclosure relates tomethods of adjusting one or more parameters in the PcPH patient toward amore normal level (e.g., normal as compared to healthy people of similarage and sex), comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1). In some embodiments, the rightventricular hypertrophy is measured using the Fulton index (RV/(LV+S)).In some embodiments, the method relates to decreasing the patient'sright ventricular hypertrophy by least 1% (e.g., 1, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). Insome embodiments, the method relates to decreasing the patient's rightventricular hypertrophy by at least 1%. In some embodiments, the methodrelates to decreasing the patient's right ventricular hypertrophy by atleast 5%. In some embodiments, the method relates to decreasing thepatient's right ventricular hypertrophy by at least 10%. In someembodiments, the method relates to decreasing the patient's rightventricular hypertrophy by at least 15%. In some embodiments, the methodrelates to decreasing the patient's right ventricular hypertrophy by atleast 20%. In some embodiments, the method relates to decreasing thepatient's right ventricular hypertrophy by at least 25%. In someembodiments, the method relates to decreasing the patient's rightventricular hypertrophy by at least 30%. In some embodiments, the methodrelates to decreasing the patient's right ventricular hypertrophy by atleast 35%. In some embodiments, the method relates to decreasing thepatient's right ventricular hypertrophy by at least 40%. In someembodiments, the method relates to decreasing the patient's rightventricular hypertrophy by at least 45%. In some embodiments, the methodrelates to decreasing the patient's right ventricular hypertrophy by atleast 50%. In some embodiments, the method relates to decreasing thepatient's right ventricular hypertrophy by at least 55%. In someembodiments, the method relates to decreasing the patient's rightventricular hypertrophy by at least 60%. In some embodiments, the methodrelates to decreasing the patient's right ventricular hypertrophy by atleast 65%. In some embodiments, the method relates to decreasing thepatient's right ventricular hypertrophy by at least 70%. In someembodiments, the method relates to decreasing the patient's rightventricular hypertrophy by at least 75%. In some embodiments, the methodrelates to decreasing the patient's right ventricular hypertrophy by atleast 80%. In some embodiments, the method relates to decreasing thepatient's right ventricular hypertrophy by at least 85%. In someembodiments, the method relates to decreasing the patient's rightventricular hypertrophy by at least 90%. In some embodiments, the methodrelates to decreasing the patient's right ventricular hypertrophy by atleast 95%. In some embodiments, the method relates to decreasing thepatient's right ventricular hypertrophy by at least 100%.

Left Ventricular Hypertrophy

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the patient has leftventricular hypertrophy. In some embodiments, the disclosure relates tomethods of adjusting one or more parameters in the PcPH patient toward amore normal level (e.g., normal as compared to healthy people of similarage and sex), comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1). In some embodiments, the methodrelates to decreasing the patient's left ventricular hypertrophy byleast 1% (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100%). In some embodiments, the methodrelates to decreasing the patient's left ventricular hypertrophy by atleast 10%. In some embodiments, the method relates to decreasing thepatient's left ventricular hypertrophy by at least 5%. In someembodiments, the method relates to decreasing the patient's leftventricular hypertrophy by at least 10%. In some embodiments, the methodrelates to decreasing the patient's left ventricular hypertrophy by atleast 15%. In some embodiments, the method relates to decreasing thepatient's left ventricular hypertrophy by at least 20%. In someembodiments, the method relates to decreasing the patient's leftventricular hypertrophy by at least 25%. In some embodiments, the methodrelates to decreasing the patient's left ventricular hypertrophy by atleast 30%. In some embodiments, the method relates to decreasing thepatient's left ventricular hypertrophy by at least 35%. In someembodiments, the method relates to decreasing the patient's leftventricular hypertrophy by at least 40%. In some embodiments, the methodrelates to decreasing the patient's left ventricular hypertrophy by atleast 45%. In some embodiments, the method relates to decreasing thepatient's left ventricular hypertrophy by at least 50%. In someembodiments, the method relates to decreasing the patient's leftventricular hypertrophy by at least 55%. In some embodiments, the methodrelates to decreasing the patient's left ventricular hypertrophy by atleast 60%. In some embodiments, the method relates to decreasing thepatient's left ventricular hypertrophy by at least 65%. In someembodiments, the method relates to decreasing the patient's leftventricular hypertrophy by at least 70%. In some embodiments, the methodrelates to decreasing the patient's left ventricular hypertrophy by atleast 75%. In some embodiments, the method relates to decreasing thepatient's left ventricular hypertrophy by at least 80%. In someembodiments, the method relates to decreasing the patient's leftventricular hypertrophy by at least 85%. In some embodiments, the methodrelates to decreasing the patient's left ventricular hypertrophy by atleast 90%. In some embodiments, the method relates to decreasing thepatient's left ventricular hypertrophy by at least 95%. In someembodiments, the method relates to decreasing the patient's leftventricular hypertrophy by at least 100%.

Cardiac Output

Cardiac output is the volume of blood the heart pumps per minute.Cardiac output is calculated by multiplying the stroke volume by theheart rate. In general, normal cardiac output at rest is about 4 to 8L/min. The cardiac index is an assessment of the cardiac output valuebased on the patient's size. To find the cardiac index, the cardiacoutput is divided by the person's body surface area (BSA). The normalrange for CI is 2.5 to 4 L/min/m². Cardiac output can decline by almost40% without deviating from the normal limits. A low cardiac index ofless than about 2.5 L/min/m² usually indicates a disturbance incardiovascular performance. The cardiac output can be utilized tocalculate the cardiac index (e.g., cardiac index=cardiac output/bodysurface area). The cardiac output can also be utilized to calculate thestroke volume (e.g., stroke volume=CO/heart rate). In certain aspects,the disclosure relates to methods of treating, preventing, or reducingthe progression rate and/or severity of PcPH (e.g., treating,preventing, or reducing the progression rate and/or severity of one ormore complications of PcPH in WHO Group 2 and/or Group 5 PH) comprisingadministering to a patient in need thereof an effective amount of anActRII polypeptide (e.g., an amino acid sequence that is at least 90%identical to an amino acid sequence corresponding to residues 30-110 ofSEQ ID NO: 1), wherein the method increases the patient's cardiac outputby at least 5% (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100%). In some embodiments, the methodrelates to increasing the patient's cardiac output by at least 5%. Insome embodiments, the method relates to increasing the patient's cardiacoutput by at least 10%. In some embodiments, the method relates toincreasing the patient's cardiac output by at least 15%. In someembodiments, the method relates to increasing the patient's cardiacoutput by at least 20%. In some embodiments, the method relates toincreasing the patient's cardiac output by at least 25%. In someembodiments, the method relates to increasing the patient's cardiacoutput by at least 30%. In some embodiments, the method relates toincreasing the patient's cardiac output by at least 35%. In someembodiments, the method relates to increasing the patient's cardiacoutput by at least 40%. In some embodiments, the method relates toincreasing the patient's cardiac output by at least 45%. In someembodiments, the method relates to increasing the patient's cardiacoutput by at least 50%. In some embodiments, the method relates toincreasing the patient's cardiac output by at least 55%. In someembodiments, the method relates to increasing the patient's cardiacoutput by at least 60%. In some embodiments, the method relates toincreasing the patient's cardiac output by at least 65%. In someembodiments, the method relates to increasing the patient's cardiacoutput by at least 70%. In some embodiments, the method relates toincreasing the patient's cardiac output by at least 75%. In someembodiments, the method relates to increasing the patient's cardiacoutput by at least 80%. In some embodiments, the method relates toincreasing the patient's cardiac output by at least 85%. In someembodiments, the method relates to increasing the patient's cardiacoutput by at least 90%. In some embodiments, the method relates toincreasing the patient's cardiac output by at least 95%. In someembodiments, the method relates to increasing the patient's cardiacoutput by at least 100%. In some embodiments, the method relates toincreasing the patient's cardiac index to at least 4.2 L/min/m². In someembodiments, the cardiac index is measured at rest. In some embodiments,the method relates to increasing the patient's cardiac output to atleast 4 L/min. In some embodiments, the cardiac output is measured atrest. In some embodiments, the cardiac output is using a right heartcatheter. In some embodiments, cardiac output is measured bythermodilution. In some embodiments, cardiac output is measured usingthe Fick method.

Progression of IpcPH to CpcPH

The predominant mechanism underlying PcPH (e.g., WHO Group 2 and/orGroup 5 PH) is elevated left-side filling pressure (i.e., left atrialpressure). Sustained elevations in left atrial pressure may causepassive pulmonary venous congestion with elevation of pulmonarypressures. In some patients, transmission of venous congestion to thepulmonary capillaries results in leakage and damage, ultimately leadingto the creation of an obstructive vasculopathy such that higherpulmonary pressures are needed to sustain forward flow. This issometimes referred to as the development of a “pre-capillary” componentof PH. In certain aspects, the disclosure relates to methods oftreating, preventing, or reducing the progression rate and/or severityof PcPH (e.g., treating, preventing, or reducing the progression rateand/or severity of one or more complications of PcPH in WHO Group 2and/or Group 5 PH) comprising administering to a patient in need thereofan effective amount of an ActRII polypeptide (e.g., an amino acidsequence that is at least 90% identical to an amino acid sequencecorresponding to residues 30-110 of SEQ ID NO: 1), wherein the methodreduces the development of a pre-capillary component of PH by at least1% (e.g., 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 1%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 2%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 3%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 4%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 5%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 10%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 15%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 20%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 25%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 30%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 35%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 40%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 45%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 50%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 55%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 60%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 65%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 70%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 75%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 80%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 85%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 90%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 95%. In someembodiments, the method relates to reducing the development of apre-capillary component of PH in a patient by at least 100%. In someembodiments, sustained left atrial pressure in IpcPH has been shown tolead to the development of CpcPH. In certain aspects, the disclosurerelates to methods of treating, preventing, or reducing the progressionrate and/or severity of PcPH (e.g., treating, preventing, or reducingthe progression rate and/or severity of one or more complications ofPcPH in WHO Group 2 and/or Group 5 PH) comprising administering to apatient in need thereof an effective amount of an ActRII polypeptide(e.g., an amino acid sequence that is at least 90% identical to an aminoacid sequence corresponding to residues 30-110 of SEQ ID NO: 1), whereinthe method reduces the progression of IpcPH to CpcPH in a patient by atleast 1% (e.g., 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In someembodiments, the method relates to reducing the progression of IpcPH toCpcPH in a patient by at least 1%. In some embodiments, the methodrelates to reducing the progression of IpcPH to CpcPH in a patient by atleast 2%. In some embodiments, the method relates to reducing theprogression of IpcPH to CpcPH in a patient by at least 3%. In someembodiments, the method relates to reducing the progression of IpcPH toCpcPH in a patient by at least 4%. In some embodiments, the methodrelates to reducing the progression of IpcPH to CpcPH in a patient by atleast 5%. In some embodiments, the method relates to reducing theprogression of IpcPH to CpcPH in a patient by at least 10%. In someembodiments, the method relates to reducing the progression of IpcPH toCpcPH in a patient by at least 15%. In some embodiments, the methodrelates to reducing the progression of IpcPH to CpcPH in a patient by atleast 20%. In some embodiments, the method relates to reducing theprogression of IpcPH to CpcPH in a patient by at least 25%. In someembodiments, the method relates to reducing the progression of IpcPH toCpcPH in a patient by at least 30%. In some embodiments, the methodrelates to reducing the progression of IpcPH to CpcPH in a patient by atleast 35%. In some embodiments, the method relates to reducing theprogression of IpcPH to CpcPH in a patient by at least 40%. In someembodiments, the method relates to reducing the progression of IpcPH toCpcPH in a patient by at least 45%. In some embodiments, the methodrelates to reducing the progression of IpcPH to CpcPH in a patient by atleast 50%. In some embodiments, the method relates to reducing theprogression of IpcPH to CpcPH in a patient by at least 55%. In someembodiments, the method relates to reducing the progression of IpcPH toCpcPH in a patient by at least 60%. In some embodiments, the methodrelates to reducing the progression of IpcPH to CpcPH in a patient by atleast 65%. In some embodiments, the method relates to reducing theprogression of IpcPH to CpcPH in a patient by at least 70%. In someembodiments, the method relates to reducing the progression of IpcPH toCpcPH in a patient by at least 75%. In some embodiments, the methodrelates to reducing the progression of IpcPH to CpcPH in a patient by atleast 80%. In some embodiments, the method relates to reducing theprogression of IpcPH to CpcPH in a patient by at least 85%. In someembodiments, the method relates to reducing the progression of IpcPH toCpcPH in a patient by at least 90%. In some embodiments, the methodrelates to reducing the progression of IpcPH to CpcPH in a patient by atleast 95%. In some embodiments, the method relates to reducing theprogression of IpcPH to CpcPH in a patient by at least 100%.

Exercise Capacity (6MWD and BDI)

In certain aspects, the disclosure relates to methods of increasingexercise capacity in a patient having PcPH (e.g., WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1). Any suitable measure of exercisecapacity can be used. For example, exercise capacity in a 6-minute walktest (6MWT), which measures how far the subject can walk in 6 minutes,i.e., the 6-minute walk distance (6MWD), is frequently used to assesspulmonary hypertension severity and disease progression. In certainaspects, the Borg dyspnea index (BDI) may be used to measure exercisecapacity. The BDI is a numerical scale for assessing perceived dyspnea(breathing discomfort). It measures the degree of breathlessness, forexample, after completion of the 6MWT, where a BDI of 0 indicates nobreathlessness and 10 indicates maximum breathlessness. In someembodiments, the BDI is measured using the BORG CR10 scale.

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., WHO Group 2 and/or Group 5 PH) comprising administering to apatient in need thereof an effective amount of an ActRII polypeptide(e.g., an amino acid sequence that is at least 90% identical to an aminoacid sequence corresponding to residues 30-110 of SEQ ID NO: 1), whereinthe patient has a 6MWD of less than 550 meters (e.g., a 6MWD of lessthan 550, 500, 450, 440, 400, 380, 350, 300, 250, 200, or 150 meters).In some embodiments, the method relates to patient's having a 6MWD ofbetween 150 to 550 meters. In some embodiments, the method relates topatient's having a 6MWD of between 100 to 500 meters. In someembodiments, the method relates to patient's having a 6MWD of between150 to 500 meters. In some embodiments, the method relates to patient'shaving a 6MWD of at least 100 meters. In some embodiments, the methodrelates to patient's having a 6MWD of greater than 150 meters. In someembodiments, the method relates to patient's having a 6MWD of less than550 meters. In some embodiments, the method relates to patient's havinga 6MWD of less than 500 meters. In some embodiments, the method relatesto patient's having a 6MWD of less than 450 meters. In some embodiments,the method relates to patient's having a 6MWD of less than 440 meters.In some embodiments, the method relates to patient's having a 6MWD ofless than 400 meters. In some embodiments, the method relates topatient's having a 6MWD of less than 380 meters. In some embodiments,the method relates to patient's having a 6MWD of less than 350 meters.In some embodiments, the method relates to patient's having a 6MWD ofless than 300 meters. In some embodiments, the method relates topatient's having a 6MWD of less than 250 meters. In some embodiments,the method relates to patient's having a 6MWD of less than 200 meters.In some embodiments, the method relates to patient's having a 6MWD ofless than 150 meters. In some embodiments, the method relates toincreasing the patient's 6MWD to >380 meters. In some embodiments, themethod relates to increasing the patient's 6MWD to >440 meters. In someembodiments, the method relates to increasing the patient's 6MWD to >500meters. See, e.g., Galie N., et al Euro Heart J. (2016) 37, 67-119.

In some embodiments, the disclosure relates to methods of adjusting oneor more measurements of exercise capacity in the PcPH (e.g., WHO Group 2and/or Group 5 PH) patient toward a more normal level (e.g., normal ascompared to healthy people of similar age and sex), comprisingadministering to a patient in need thereof an effective amount of anActRII polypeptide (e.g., an amino acid sequence that is at least 90%identical to an amino acid sequence corresponding to residues 30-110 ofSEQ ID NO: 1). In some embodiments, the method relates to increasing thepatient's 6MWD by at least 10 meters. In some embodiments, the methodrelates to increasing the patient's 6MWD by at least 20 meters. In someembodiments, the method relates to increasing the patient's 6MWD by atleast 25 meters. In some embodiments, the method relates to increasingthe patient's 6MWD by at least 30 meters. In some embodiments, themethod relates to increasing the patient's 6MWD by at least 40 meters.In some embodiments, the method relates to increasing the patient's 6MWDby at least 50 meters. In some embodiments, the method relates toincreasing the patient's 6MWD by at least 60 meters. In someembodiments, the method relates to increasing the patient's 6MWD by atleast 70 meters. In some embodiments, the method relates to increasingthe patient's 6MWD by at least 80 meters. In some embodiments, themethod relates to increasing the patient's 6MWD by at least 90 meters.In some embodiments, the method relates to increasing the patient's 6MWDby at least 100. In some embodiments, the method relates to increasingthe patient's 6MWD by at least 125. In some embodiments, the methodrelates to increasing the patient's 6MWD by at least 150 meters In someembodiments, the method relates to increasing the patient's 6MWD by atleast 175 meters. In some embodiments, the method relates to increasingthe patient's 6MWD by at least 200 meters. In some embodiments, themethod relates to increasing the patient's 6MWD by at least 250 meters.In some embodiments, the method relates to increasing the patient's 6MWDby at least 300 meters. In some embodiments, the method relates toincreasing the patient's 6MWD by at least 400 meters. In someembodiments, the 6MWD is tested after the patient has received 4 weeksof treatment utilizing an ActRII polypeptide disclosed herein. In someembodiments, the 6MWD is tested after the patient has received 8 weeksof treatment utilizing an ActRII polypeptide disclosed herein. In someembodiments, the 6MWD is tested after the patient has received 12 weeksof treatment utilizing an ActRII polypeptide disclosed herein. In someembodiments, the 6MWD is tested after the patient has received 16 weeksof treatment utilizing an ActRII polypeptide disclosed herein. In someembodiments, the 6MWD is tested after the patient has received 20 weeksof treatment utilizing an ActRII polypeptide disclosed herein. In someembodiments, the 6MWD is tested after the patient has received 22 weeksof treatment utilizing an ActRII polypeptide disclosed herein. In someembodiments, the 6MWD is tested after the patient has received 24 weeksof treatment utilizing an ActRII polypeptide disclosed herein. In someembodiments, the 6MWD is tested after the patient has received 26 weeksof treatment utilizing an ActRII polypeptide disclosed herein. In someembodiments, the 6MWD is tested after the patient has received 28 weeksof treatment utilizing an ActRII polypeptide disclosed herein. In someembodiments, the 6MWD is tested after the patient has received 48 weeksof treatment utilizing an ActRII polypeptide disclosed herein.

In some embodiments, the disclosure relates to methods of adjusting oneor more measurements of exercise capacity (e.g., BDI) in the PcPH (e.g.,WHO Group 2 and/or Group 5 PH) patient toward amore normal level (e.g.,normal as compared to healthy people of similar age and sex), comprisingadministering to a patient in need thereof an effective amount of anActRII polypeptide (e.g., an amino acid sequence that is at least 90%identical to an amino acid sequence corresponding to residues 30-110 ofSEQ ID NO: 1). In some embodiments, the method relates to reducing thepatient's BDI. In some embodiments, the method relates to lowering thepatient's BDI by at least 0.5 index points. In some embodiments, themethod relates to lowering the patient's BDI by at least 1 index points.In some embodiments, the method relates to lowering the patient's BDI byat least 1.5 index points. In some embodiments, the method relates tolowering the patient's BDI by at least 2 index points. In someembodiments, the method relates to lowering the patient's BDI by atleast 2.5 index points In some embodiments, the method relates tolowering the patient's BDI by at least 3 index points. In someembodiments, the method relates to lowering the patient's BDI by atleast 3.5 index points. In some embodiments, the method relates tolowering the patient's BDI by at least 4 index points. In someembodiments, the method relates to lowering the patient's BDI by atleast 4.5 index points. In some embodiments, the method relates tolowering the patient's BDI by at least 5 index points. In someembodiments, the method relates to lowering the patient's BDI by atleast 5.5 index points. In some embodiments, the method relates tolowering the patient's BDI by at least 6 index points. In someembodiments, the method relates to lowering the patient's BDI by atleast 6.5 index points. In some embodiments, the method relates tolowering the patient's BDI by at least 7 index points. In someembodiments, the method relates to lowering the patient's BDI by atleast 7.5 index points. In some embodiments, the method relates tolowering the patient's BDI by at least 8 index points. In someembodiments, the method relates to lowering the patient's BDI by atleast 8.5 index points. In some embodiments, the method relates tolowering the patient's BDI by at least 9 index points. In someembodiments, the method relates to lowering the patient's BDI by atleast 9.5 index points. In some embodiments, the method relates tolowering the patient's BDI by at least 3 index points. In someembodiments, the method relates to lowering the patient's BDI by atleast 10 index points.

Echocardiography

There are numerous clinical presentation factors, echocardiographyfeatures, and other features that could be indicative of PcPH (e.g., WHOGroup 2 and/or Group 5 PH). For instance, patients who are ≥65 years oldare at higher risk for WHO Group 2 (also known as PH-LHD). In patientssuspected of having PcPH (e.g., WHO Group 2 and/or Group 5 PH), anechocardiogram may be used to image the effects of PH on the heart andestimate the mPAP from continuous wave Doppler measurements. In someembodiments, an echocardiogram performed on a patient shows structuralleft heart abnormalities. In some embodiments, the structural left heartabnormality is a disease of the left heart valves. In some embodiments,the structural left heart abnormality is left atrium enlargement(e.g., >4.2 cm). In some embodiments, an electrocardiogram performed ona patient shows left ventricular hypertrophy (LVH) and/or left atrialhypertrophy/dilation (LAH). In some embodiments, an electrocardiogramperformed on a patient shows atrial flutter/atrial fibrillation(AF/Afib). In some embodiments, an electrocardiogram performed on apatient shows left bundle branch block (LBBB). In some embodiments, anelectrocardiogram performed on a patient shows the presence of Q waves.See, e.g., Galie N., et al Euro Heart J. (2016) 37, 67-119.

In a patient that has symptoms of left heart failure, an echocardiogrammay be performed to evaluate various parameters. For instance, in someembodiments, an echocardiogram using Doppler performed on a patient mayshow indices of increased filling pressures and/or diastolic dysfunction(e.g., increased E/E′ or >Type 2-3 mitral flow abnormality). In someembodiments, imaging (e.g. echocardiogram, CT scan, chest X-ray, or MRI)performed on a patient shows Kerley B lines. In some embodiments,imaging (e.g. echocardiogram, CT scan, chest X-ray, or MRI) performed ona patient shows pleural effusion. In some embodiments, imaging (e.g.echocardiogram, CT scan, chest X-ray, or MRI) performed on a patientshows pulmonary edema. In some embodiments, imaging (e.g.,echocardiogram, CT scan, chest X-ray, or MRI) performed on a patientshows left atrium enlargement. Id.

Furthermore, in a patient that has features of metabolic syndrome,imaging (e.g. an echocardiogram) may be performed to evaluate variousparameters. For instance, in some embodiments, an echocardiogramperformed on a patient shows the absence of right ventricle dysfunction(e.g., IpcPH). In some embodiments, an echocardiogram performed on apatient shows the presence of right ventricle dysfunction (e.g., CpcPH).In some embodiments, an echocardiogram performed on a patient shows theabsence of mid systolic notching of the pulmonary artery flow. In someembodiments, an echocardiogram performed on a patient shows the absenceof pericardial effusion. In some embodiments, the patient has a historyof heart disease (past or current). In some embodiments, the patient haspersistent atrial fibrillation. Id. In some embodiments, an Echo Scoreor the TAPSE/systolic pulmonary arterial pressure ratio are used todifferentiate Cpc-PH from Ipc-PH. In some embodiments, an integrativescore of five echocardiographic parameters (RV/LV ratio, leftventricular eccentricity index (LVEI), E/E′, RV forming apex, width andinspiratory collapse of IVC) as well as “notching” of the RV outflowtract Doppler envelope may be used to distinguish between precapillaryPH (e.g., PAH) from post-capillary PH (e.g., WHO Group 2 and/or Group 5PH).

In some embodiments, a patient has diastolic dysfunction. In someembodiments, the method improves diastolic dysfunction in the patient.In some embodiments, the improvement in diastolic dysfunction is animprovement in the E/E′ ratio (a ratio of mitral inflow velocity (E) tomitral annular velocity (E′). In some embodiments, the improvement indiastolic dysfunction is an improvement in the isovolumic relaxationtime (IVRT). In some embodiments, the improvement in the diastolicdysfunction is a lower RVSP. In some embodiments, the diastolicdysfunction results from one or more conditions selected from the groupconsisting of hypertension, diabetes, and advanced age.

Complications of PH

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of one ormore complications of PcPH (e.g., WHO Group 2 and/or Group 5 PH)comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the method relates totreating, preventing, or reducing the progression rate and/or severityof cell proliferation in the pulmonary artery of a PcPH patient. In someembodiments, the method relates to treating, preventing, or reducing theprogression rate and/or severity of smooth muscle and/or endothelialcells proliferation in the pulmonary artery of a PcPH patient. In someembodiments, the method relates to treating, preventing, or reducing theprogression rate and/or severity of angiogenesis in the pulmonary arteryof a PcPH patient. In some embodiments, the method relates to increasingphysical activity of a patient having PcPH. In some embodiments, themethod relates to treating, preventing, or reducing the progression rateand/or severity of dyspnea in a PcPH patient. In some embodiments, themethod relates to treating, preventing, or reducing the progression rateand/or severity of chest pain in a PcPH patient. In some embodiments,the method relates to treating, preventing, or reducing the progressionrate and/or severity of fatigue in a PcPH patient. In some embodiments,the method relates to treating, preventing, or reducing the progressionrate and/or severity of fibrosis in a PcPH patient. In some embodiments,the fibrosis is selected from the group consisting of left ventricularfibrosis, right ventricular fibrosis, and pulmonary fibrosis. In someembodiments, the method relates to treating, preventing, or reducing theprogression rate and/or severity of left ventricular fibrosis in a PcPHpatient. In some embodiments, the method relates to treating,preventing, or reducing the progression rate and/or severity of rightventricular fibrosis in a PcPH patient. In some embodiments, the methodrelates to treating, preventing, or reducing the progression rate and/orseverity of pulmonary fibrosis in a PcPH patient. In some embodiments,the method relates to decreasing the patient's fibrosis by least 1%(e.g., 1%, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, or 100%). In some embodiments, the method relates todecreasing the patient's fibrosis by at least 1%. In some embodiments,the method relates to decreasing the patient's fibrosis by at least 5%.In some embodiments, the method relates to decreasing the patient'sfibrosis by at least 10%. In some embodiments, the method relates todecreasing the patient's fibrosis by at least 15%. In some embodiments,the method relates to decreasing the patient's fibrosis by at least 20%.In some embodiments, the method relates to decreasing the patient'sfibrosis by at least 25%. In some embodiments, the method relates todecreasing the patient's fibrosis by at least 30%. In some embodiments,the method relates to decreasing the patient's fibrosis by at least 35%.In some embodiments, the method relates to decreasing the patient'sfibrosis by at least 40%. In some embodiments, the method relates todecreasing the patient's fibrosis by at least 45%. In some embodiments,the method relates to decreasing the patient's fibrosis by at least 50%.In some embodiments, the method relates to decreasing the patient'sfibrosis by at least 55%. In some embodiments, the method relates todecreasing the patient's fibrosis by at least 60%. In some embodiments,the method relates to decreasing the patient's fibrosis by at least 65%.In some embodiments, the method relates to decreasing the patient'sfibrosis by at least 70%. In some embodiments, the method relates todecreasing the patient's fibrosis by at least 75%. In some embodiments,the method relates to decreasing the patient's fibrosis by at least 80%.In some embodiments, the method relates to decreasing the patient'sfibrosis by at least 85%. In some embodiments, the method relates todecreasing the patient's fibrosis by at least 90%. In some embodiments,the method relates to decreasing the patient's fibrosis by at least 95%.In some embodiments, the method relates to decreasing the patient'sfibrosis by at least 100%.

In some embodiments, the method relates to treating, preventing, orreducing the progression rate and/or severity of pulmonary vascularremodeling in a PcPH patient. In some embodiments, the method relates totreating, preventing, or reducing the progression rate and/or severityof cardiac remodeling in a PcPH patient. In some embodiments, the methodrelates to treating, preventing, or reducing the progression rate and/orseverity of left cardiac remodeling in a PcPH patient. In someembodiments, the method relates to treating, preventing, or reducing theprogression rate and/or severity of right cardiac remodeling in a PcPHpatient. In some embodiments, the method relates to treating,preventing, or reducing the progression rate and/or severity of rightventricular hypertrophy in a PcPH patient. In some embodiments, themethod relates to treating, preventing, or reducing the progression rateand/or severity of left ventricular hypertrophy in a PcPH patient Insome embodiments, the method relates to treating, preventing, orreducing the progression rate and/or severity of metabolic syndrome in aPcPH patient. In some embodiments, the method relates to treating,preventing, or reducing the progression rate and/or severity of leftatrium dilation in a PcPH patient. In some embodiments, the methodrelates to treating, preventing, or reducing the progression rate and/orseverity of an underlying condition (e.g., COPD, sleep apnea syndrome,CTEPH) in a PcPH patient.

Complications or Comorbidities and Combination Therapies

In some embodiments, the disclosure contemplates methods of treating oneor more complications of PcPH (e.g., smooth muscle and/or endothelialcell proliferation in the pulmonary artery, angiogenesis in thepulmonary artery, dyspnea, chest pain, pulmonary vascular remodeling,cardiac remodeling, right ventricular hypertrophy, left ventricularhypertrophy, left atrium dilation, pulmonary fibrosis, need for lungand/or heart transplant, and need for atrial septostomy) comprisingadministering to a patient in need thereof an effective amount of anActRII polypeptide (e.g., an amino acid sequence that is at least 90%identical to an amino acid sequence corresponding to residues 30-110 ofSEQ ID NO: 1). In some embodiments, the disclosure contemplates methodsof preventing one or more complications of PcPH comprising administeringto a patient in need thereof an effective amount of an ActRIIpolypeptide (e.g., an amino acid sequence that is at least 90% identicalto an amino acid sequence corresponding to residues 30-110 of SEQ ID NO:1). In some embodiments, the disclosure contemplates methods of reducingthe progression rate of one or more complications of PcPH comprisingadministering to a patient in need thereof an effective amount of anActRII polypeptide (e.g., an amino acid sequence that is at least 90%identical to an amino acid sequence corresponding to residues 30-110 ofSEQ ID NO: 1). In some embodiments, the disclosure contemplates methodsof reducing the severity of one or more complications of PcPH comprisingadministering to a patient in need thereof an effective amount of anActRII polypeptide (e.g., an amino acid sequence that is at least 90%identical to an amino acid sequence corresponding to residues 30-110 ofSEQ ID NO: 1).

In some embodiments, the disclosure contemplates methods of treating oneor more comorbidities of PcPH (e.g., systemic hypertension, decreasedrenal function, diabetes mellitus, obesity, coronary artery disease(CAD), heart failure, and anemia) comprising administering to a patientin need thereof an effective amount of an ActRII polypeptide (e.g., anamino acid sequence that is at least 90% identical to an amino acidsequence corresponding to residues 30-110 of SEQ ID NO: 1). In someembodiments, the method results in the improvement of one or morecomorbidities of PcPH (e.g., systemic hypertension, decreased renalfunction, diabetes mellitus, obesity, coronary artery disease (CAD),heart failure, and anemia). In some embodiments, the one or morecomorbidities of PcPH are improved indirectly (e.g., due to animprovement in the patient's PH).

In some embodiments, the disclosure contemplates methods of reducing theprogression rate of PcPH (e.g., WHO Group 2 and/or Group 5 PH)comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the disclosurecontemplates methods of reducing the progression rate of one or morecomplications of PcPH (e.g., WHO Group 2 and/or Group 5 PH) comprisingadministering to a patient in need thereof an effective amount of anActRII polypeptide (e.g., an amino acid sequence that is at least 90%identical to an amino acid sequence corresponding to residues 30-110 ofSEQ ID NO: 1). In some embodiments, the disclosure contemplates methodsof reducing the severity of PcPH (e.g., WHO Group 2 and/or Group 5 PH)comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the disclosurecontemplates methods of reducing the severity of one or morecomplications of PcPH (e.g., WHO Group 2 and/or Group 5 PH) comprisingadministering to a patient in need thereof an effective amount of anActRII polypeptide (e.g., an amino acid sequence that is at least 90%identical to an amino acid sequence corresponding to residues 30-110 ofSEQ ID NO: 1). In some embodiments, the disclosure contemplates methodof reducing the need to initiate treatment with a known treatment forPcPH comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the disclosurecontemplates a method of reducing the need to increase the dose ofprostacyclin in a patient (e.g., increasing the dose by at least 10%)comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1). In some embodiments, the disclosurecontemplates a method of reducing the need for PcPH-specifichospitalization comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1). In some embodiments, PcPH-specifichospitalization is hospitalization of patient for at least 24 hours. Insome embodiments, the disclosure contemplates a method of reducing thedeterioration of PcPH comprising administering to a patient in needthereof an effective amount of an ActRII polypeptide (e.g., an aminoacid sequence that is at least 90% identical to an amino acid sequencecorresponding to residues 30-110 of SEQ ID NO: 1). In some embodiments,deterioration of PcPH comprises worsening in WHO functional class and/ora decrease of at least 15% in the 6MWD of the patient.

Optionally, methods disclosed herein for treating, preventing, orreducing the progression rate and/or severity of PcPH (e.g., WHO Group 2and/or Group 5 PH), particularly treating, preventing, or reducing theprogression rate and/or severity of one or more complications of PcPH,may further comprise administering to the patient one or more supportivetherapies or additional active agents for treating PcPH. For example,the patient also may be administered one or more supportive therapies oractive agents selected from the group consisting of: nitrates,hydralazine, prostacyclin and derivatives thereof (e.g., epoprostenol,treprostinil, and iloprost); prostacyclin receptor agonists (e.g.,selexipag); endothelin receptor antagonists (e.g., thelin, ambrisentan,macitentan, darusentan, and bosentan); calcium channel blockers (e.g.,amlodipine, diltiazem, and nifedipine; anticoagulants (e.g., warfarin);diuretics; oxygen therapy; atrial septostomy; pulmonarythromboendarterectomy; phosphodiesterase type 5 inhibitors (e.g.,sildenafil and tadalafil); activators of soluble guanylate cyclase(e.g., cinaciguat, vericiguat, and riociguat); ASK-1 inhibitors (e.g.,CIIA; SCH79797; GS-4997; MSC2032964A;3H-naphtho[1,2,3-de]quiniline-2,7-diones, NQDI-1;2-thioxo-thiazolidines,5-bromo-3-(4-oxo-2-thioxo-thiazolidine-5-ylidene)-1,3-dihydro-indol-2-one);NF-κB antagonists (e.g., dh404, CDDO-epoxide; 2,2-difluoropropionamide;C28 imidazole (CDDO-Im); 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid(CDDO); 3-Acetyloleanolic Acid; 3-Triflouroacetyloleanolic Acid;28-Methyl-3-acetyloleanane; 28-Methyl-3-trifluoroacetyloleanane;28-Methyloxyoleanolic Acid; SZC014; SCZ015; SZC017; PEGylatedderivatives of oleanolic acid; 3-O-(beta-D-glucopyranosyl) oleanolicacid; 3-O-[beta-D-glucopyranosyl-(1→3)-beta-D-glucopyranosyl] oleanolicacid; 3-0-[beta-D-glucopyranosyl-(1→2)-beta-D-glucopyranosyl] oleanolicacid; 3-O-[beta-D-glucopyranosyl-(1→3)-beta-D-glucopyranosyl] oleanolicacid 28-O-beta-D-glucopyranosyl ester;3-O-[beta-D-glucopyranosyl-(1→2)-beta-D-glucopyranosyl] oleanolic acid28-O-beta-D-glucopyranosyl ester;3-O-[a-L-rhamnopyranosyl-(1→3)-beta-D-glucuronopyranosyl]oleanolic acid;3-O-[alpha-L-rhamnopyranosyl-(1→3)-beta-D-glucuronopyranosyl] oleanolicacid 28-O-beta-D-glucopyranosyl ester; 28-O-β-D-glucopyranosyl-oleanolicacid; 3-O-p-D-glucopyranosyl (1→3)-β-D-glucopyranosiduronic acid (CS1);oleanolic acid 3-O-p-D-glucopyranosyl (1→3)-β-D-glucopyranosiduronicacid (CS2); methyl 3,11-dioxoolean-12-en-28-olate (DIOXOL); ZCVI₄-2;Benzyl 3-dehydr-oxy-1,2,5-oxadiazolo[3′,4′:2,3]oleanolate), an LV assistdevice (LVAD), implantable cardioverter-defibrillator (ICD), valvereplacement, valve repair, lung and/or heart transplantation. In someembodiments, the methods described herein may further compriseadministering to the patient parental prostacyclin. In some embodiments,the methods described herein may further comprise administering to thepatient one additional supportive therapy or additional active agent(i.e., double therapy) for treating PcPH. In some embodiments, themethods described herein may further comprise administering to thepatient two additional supportive therapies or additional active agents(i.e., triple therapy) for treating PcPH. In some embodiments, themethods described herein may further comprise administering to thepatient three additional supportive therapies or additional activeagents (i.e., quadruple therapy) for treating PcPH.

In some embodiments, the methods described herein may further compriseadministering to the patient an angiotensin antagonist (e.g.,angiotensin receptor blocker, ARB). In some embodiments, a patient isfurther administered one or more ARBs selected from the group consistingof losartan, irbesartan, olmesartan, candesartan, valsartan, fimasartan,azilsartan, salprisartan, and telmisartan. In some embodiments, apatient is administered losartan. In some embodiments, a patient isadministered irbesartan. In some embodiments, a patient is administeredolmesartan. In some embodiments, a patient is administered candesartan.In some embodiments, a patient is administered valsartan. In someembodiments, a patient is administered fimasartan. In some embodiments,a patient is administered azilsartan. In some embodiments, a patient isadministered salprisartan. In some embodiments, a patient isadministered telmisartan. In some embodiments, the methods describedherein may further comprise administering to the patient a neprilysininhibitor and an angiotensin antagonist (e.g., sacubitril/valsartan;also known as LCZ696).

In some embodiments, the methods described herein may further compriseadministering to the patient one or more ACE inhibitors. In someembodiments, the one or more ACE inhibitors are selected from the groupconsisting of benazepril, captopril, enalapril, lisinopril, perindopril,ramipril (e.g., ramipen), trandolapril, and zofenopril. In someembodiments, a patient is administered benazepril. In some embodiments,a patient is administered captopril. In some embodiments, a patient isadministered enalapril. In some embodiments, a patient is administeredlisinopril. In some embodiments, a patient is administered perindopril.In some embodiments, a patient is administered ramipril. In someembodiments, a patient is administered trandolapril. In someembodiments, a patient is administered zofenopril. In some embodiments,the methods described herein may further comprise administering to thepatient an ARB and an ACE inhibitor. In some embodiments, an alternativeapproach to angiotensin antagonism is to combine an ACE inhibitor and/orARB with an aldosterone antagonist.

In some embodiments, the methods described herein may further compriseadministering to the patient one or more neprilysin inhibitors. In someembodiments, the one or more neprilysin inhibitors are selected from thegroup consisting of thiorphan, phosphoramidon, candoxatrilat,candoxatril, ecadotril, omapatrilat, LBQ657, and sacubitril. In someembodiments, a patient is administered thiorphan. In some embodiments, apatient is administered phosphoramidon. In some embodiments, a patientis administered candoxatrilat. In some embodiments, a patient isadministered candoxatril. In some embodiments, a patient is administeredecadotril. In some embodiments, a patient is administered omapatrilat.In some embodiments, a patient is administered LBQ657. In someembodiments, a patient is administered sacubitril. In some embodiments,the methods described herein may further comprise administering to thepatient a neprilysin inhibitor and an ARB (e.g., sacubitril/valsartan;also known as LCZ696).

In some embodiments, the methods described herein may further compriseadministering to the patient an angiotensin receptor-neprilysininhibitor (ARNI). In some embodiments, the ARNI is sacubitril/valsartan(Entresto®). In some embodiments, a patient is administeredsacubitril/valsartan (Entresto®).

In some embodiments, the methods described herein may further compriseadministering to the patient one or more beta-blockers. In someembodiments, the one or more beta-blockers are selected from the groupconsisting of bisoprolol, carvedilol, metoprolol succinate (CR/XL), andnebivolol. In some embodiments, a patient is administered bisoprolol. Insome embodiments, a patient is administered carvedilol. In someembodiments, a patient is administered metoprolol succinate (CR/XL). Insome embodiments, a patient is administered nebivolol.

In some embodiments, the methods described herein may further compriseadministering to the patient one or more mineralocorticoid receptorantagonists (MRA). In some embodiments, the one or more MRA are selectedfrom the group consisting of eplerenone and spironolactone. In someembodiments, a patient is administered eplerenone. In some embodiments,a patient is administered spironolactone.

In some embodiments, the methods described herein may further compriseadministering to the patient one or more hyperpolarization-activatedcyclic nucleotide-gated (HCN) channel blockers. In some embodiments, theone or more HCN are selected from the group consisting of ivabradine,ZD7288, cilobradine, zatebradine, alinidine, clonidine, falipamil,TH92:20, and YM758. In some embodiments, a patient is administeredivabradine. In some embodiments, a patient is administered ZD7288. Insome embodiments, a patient is administered cilobradine. In someembodiments, a patient is administered zatebradine. In some embodiments,a patient is administered alinidine. In some embodiments, a patient isadministered clonidine. In some embodiments, a patient is administeredfalipamil. In some embodiments, a patient is administered TH92:20. Insome embodiments, a patient is administered YM758.

In some embodiments, the one or more supportive therapies or additionalactive agents for treating PcPH are administered prior to administrationof the ActRII polypeptide. In some embodiments, the one or moresupportive therapies or additional active agents for treating PcPH areadministered in combination with the ActRII polypeptide. In someembodiments, the one or more supportive therapies or additional activeagents for treating PcPH are administered after the administration ofthe ActRII polypeptide. As used herein, a therapeutic that “prevents” adisorder or condition refers to a compound that, in a statisticalsample, reduces the occurrence of the disorder or condition in thetreated sample relative to an untreated control sample, or delays theonset or reduces the severity of one or more symptoms of the disorder orcondition relative to the untreated control sample.

Transplant Free Survival

Lung and/or heart transplantation is a surgical treatment option forpatients with PcPH, and is often recommended for patients who don'trespond to less invasive therapies (e.g., vasodilator therapy).Generally, PcPH patients who receive lung and/or heart transplantationhave Functional Class III or Class IV pulmonary hypertension inaccordance with the World Health Organization's functionalclassification system for pulmonary hypertension.

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPHcomprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide (e.g., an amino acid sequence that is atleast 90% identical to an amino acid sequence corresponding to residues30-110 of SEQ ID NO: 1), wherein the method increases the patient'stransplant free survival by at least 1% (e.g., 1%, 2%, 3%, 4%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%. In some embodiments, the method relates toincreasing the patient's transplant free survival by at least 1%. Insome embodiments, the method relates to increasing the patient'stransplant free survival by at least 2%. In some embodiments, the methodrelates to increasing the patient's transplant free survival by at least3%. In some embodiments, the method relates to increasing the patient'stransplant free survival by at least 4%. In some embodiments, the methodrelates to increasing the patient's transplant free survival by at least5%. In some embodiments, the method relates to increasing the patient'stransplant free survival by at least 10%. In some embodiments, themethod relates to increasing the patient's transplant free survival byat least 15%. In some embodiments, the method relates to increasing thepatient's transplant free survival by at least 20%. In some embodiments,the method relates to increasing the patient's transplant free survivalby at least 25%. In some embodiments, the method relates to increasingthe patient's transplant free survival by at least 30%. In someembodiments, the method relates to increasing the patient's transplantfree survival by at least 35%. In some embodiments, the method relatesto increasing the patient's transplant free survival by at least 40%. Insome embodiments, the method relates to increasing the patient'stransplant free survival by at least 45%. In some embodiments, themethod relates to increasing the patient's transplant free survival byat least 50%. In some embodiments, the method relates to increasing thepatient's transplant free survival by at least 55%. In some embodiments,the method relates to increasing the patient's transplant free survivalby at least 60%. In some embodiments, the method relates to increasingthe patient's transplant free survival by at least 65%. In someembodiments, the method relates to increasing the patient's transplantfree survival by at least 70%. In some embodiments, the method relatesto increasing the patient's transplant free survival by at least 75%. Insome embodiments, the method relates to increasing the patient'stransplant free survival by at least 80%. In some embodiments, themethod relates to increasing the patient's transplant free survival byat least 85%. In some embodiments, the method relates to increasing thepatient's transplant free survival by at least 90%. In some embodiments,the method relates to increasing the patient's transplant free survivalby at least 95%. In some embodiments, the method relates to increasingthe patient's transplant free survival by at least 100%. In someembodiments, the method relates to increasing the patient's transplantfree survival as compared to controls over 1 year. In some embodiments,the method relates to increasing the patient's transplant free survivalas compared to controls over 2 years. In some embodiments, the methodrelates to increasing the patient's transplant free survival as comparedto controls over 3 years. In some embodiments, the method relates toincreasing the patient's transplant free survival as compared tocontrols over 4 years. In some embodiments, the method relates toincreasing the patient's transplant free survival as compared tocontrols over 5 years. In some embodiments, the method relates toincreasing the patient's transplant free survival as compared tocontrols over 6 years. In some embodiments, the method relates toincreasing the patient's transplant free survival as compared tocontrols over 7 years.

Death

In certain aspects, the disclosure relates to methods of reducing therisk of death in patients with PcPH comprising administering to apatient in need thereof an effective amount of an ActRII polypeptide(e.g., an amino acid sequence that is at least 90% identical to an aminoacid sequence corresponding to residues 30-110 of SEQ ID NO: 1), whereinthe method reduces the patient's risk of death by at least 1% (e.g., 1%,2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%). In some embodiments, themethod relates to reducing the patient's risk of death by at least 1%.In some embodiments, the method relates to reducing the patient's riskof death by at least 2%. In some embodiments, the method relates toreducing the patient's risk of death by at least 3%. In someembodiments, the method relates to reducing the patient's risk of deathby at least 4%. In some embodiments, the method relates to reducing thepatient's risk of death by at least 5%. In some embodiments, the methodrelates to reducing the patient's risk of death by at least 10%. In someembodiments, the method relates to reducing the patient's risk of deathby at least 15%. In some embodiments, the method relates to reducing thepatient's risk of death by at least 20%. In some embodiments, the methodrelates to reducing the patient's risk of death by at least 25%. In someembodiments, the method relates to reducing the patient's risk of deathby at least 30%. In some embodiments, the method relates to reducing thepatient's risk of death by at least 35%. In some embodiments, the methodrelates to reducing the patient's risk of death by at least 40%. In someembodiments, the method relates to reducing the patient's risk of deathby at least 45%. In some embodiments, the method relates to reducing thepatient's risk of death by at least 50%. In some embodiments, the methodrelates to reducing the patient's risk of death by at least 55%. In someembodiments, the method relates to reducing the patient's risk of deathby at least 60%. In some embodiments, the method relates to reducing thepatient's risk of death by at least 65%. In some embodiments, the methodrelates to reducing the patient's risk of death by at least 70%. In someembodiments, the method relates to reducing the patient's risk of deathby at least 75%. In some embodiments, the method relates to reducing thepatient's risk of death by at least 80%. In some embodiments, the methodrelates to reducing the patient's risk of death by at least 85%. In someembodiments, the method relates to reducing the patient's risk of deathby at least 90%. In some embodiments, the method relates to reducing thepatient's risk of death by at least 95%. In some embodiments, the methodrelates to reducing the patient's risk of death by at least 100%. Insome embodiments, the method reduces the risk of hospitalization for oneor more complications associated with PcPH.

Composite Clinical Endpoint

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the method reduces thepatient's composite clinical endpoint by at least 1% (e.g., 1%, 2%, 3%,4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100%). In some embodiments, the methodrelates to reducing the patient's composite clinical endpoint by atleast 1%. In some embodiments, the method relates to reducing thepatient's composite clinical endpoint by at least 2%. In someembodiments, the method relates to reducing the patient's compositeclinical endpoint by at least 3%. In some embodiments, the methodrelates to reducing the patient's composite clinical endpoint by atleast 4%. In some embodiments, the method relates to reducing thepatient's composite clinical endpoint by at least 5%. In someembodiments, the method relates to reducing the patient's compositeclinical endpoint by at least 10%. In some embodiments, the methodrelates to reducing the patient's composite clinical endpoint by atleast 15%. In some embodiments, the method relates to reducing thepatient's composite clinical endpoint by at least 20%. In someembodiments, the method relates to reducing the patient's compositeclinical endpoint by at least 25%. In some embodiments, the methodrelates to reducing the patient's composite clinical endpoint by atleast 30%. In some embodiments, the method relates to reducing thepatient's composite clinical endpoint by at least 35%. In someembodiments, the method relates to reducing the patient's compositeclinical endpoint by at least 40%. In some embodiments, the methodrelates to reducing the patient's composite clinical endpoint by atleast 45%. In some embodiments, the method relates to reducing thepatient's composite clinical endpoint by at least 50%. In someembodiments, the method relates to reducing the patient's compositeclinical endpoint by at least 55%. In some embodiments, the methodrelates to reducing the patient's composite clinical endpoint by atleast 60%. In some embodiments, the method relates to reducing thepatient's composite clinical endpoint by at least 65%. In someembodiments, the method relates to reducing the patient's compositeclinical endpoint by at least 70%. In some embodiments, the methodrelates to reducing the patient's composite clinical endpoint by atleast 75%. In some embodiments, the method relates to reducing thepatient's composite clinical endpoint by at least 80%. In someembodiments, the method relates to reducing the patient's compositeclinical endpoint by at least 85%. In some embodiments, the methodrelates to reducing the patient's composite clinical endpoint by atleast 90%. In some embodiments, the method relates to reducing thepatient's composite clinical endpoint by at least 95%. In someembodiments, the method relates to reducing the patient's compositeclinical endpoint by at least 100%. In some embodiments, the methodreduces the risk of hospitalization for one or more complicationsassociated with PcPH. In some embodiments, the composite clinicalendpoint comprises one or more endpoints selected from the groupconsisting of clinical worsening, hospitalization, and death.

Functional Classes

PcPH (e.g., WHO Group 2 and Group 5 PH) at baseline can be mild,moderate or severe, as measured for example by World Health Organization(WHO) functional class, which is a measure of disease severity inpatients with pulmonary hypertension. The WHO functional classificationis an adaptation of the New York Heart Association (NYHA) system and isroutinely used to qualitatively assess activity tolerance, for examplein monitoring disease progression and response to treatment (Rubin(2004) Chest 126:7-10). Four functional classes are recognized in theWHO system: Class I: pulmonary hypertension without resulting limitationof physical activity; ordinary physical activity does not cause unduedyspnea or fatigue, chest pain or near syncope; Class II: pulmonaryhypertension resulting in slight limitation of physical activity;patient comfortable at rest; ordinary physical activity causes unduedyspnea or fatigue, chest pain or near syncope; Class III: pulmonaryhypertension resulting in marked limitation of physical activity;patient comfortable at rest; less than ordinary activity causes unduedyspnea or fatigue, chest pain or near syncope; Class IV: pulmonaryhypertension resulting in inability to carry out any physical activitywithout symptoms; patient manifests signs of right-heart failure;dyspnea and/or fatigue may be present even at rest; discomfort isincreased by any physical activity.

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the patient has FunctionalClass I, Functional Class II, Functional Class III, or Functional ClassIV pulmonary hypertension as recognized by the WHO. In some embodiments,the method relates to a patient that has Functional Class I pulmonaryhypertension as recognized by the WHO. In some embodiments, the methodrelates to a patient that has Functional Class II pulmonary hypertensionas recognized by the WHO. In some embodiments, the method relates to apatient that has Functional Class III pulmonary hypertension asrecognized by the WHO. In some embodiments, the method relates to apatient that has Functional Class IV pulmonary hypertension asrecognized by the WHO. In some embodiments, the method relates topatients having Functional Class II or Class III pulmonary hypertensionas recognized by the WHO. In some embodiments, the method relates topatients having Functional Class II, Class III, or Class IV pulmonaryhypertension as recognized by the WHO. In some embodiments, the methodrelates to patients having Functional Class I, Class II, Class III, orClass IV pulmonary hypertension as recognized by the WHO. In someembodiments, the method delays clinical worsening of PcPH. In someembodiments, the method delays clinical worsening of PcPH in accordancewith the WHO's functional classification system for pulmonaryhypertension.

In some embodiments, the disclosure relates to methods of preventing orreducing pulmonary hypertension Functional Class progression comprisingadministering to a patient in need thereof an effective amount of anActRII polypeptide (e.g., an amino acid sequence that is at least 90%identical to an amino acid sequence corresponding to residues 30-110 ofSEQ ID NO: 1). In some embodiments, the reduction in Functional Classprogression is a delay in Functional Class progression. In someembodiments, the method relates to preventing or decreasing pulmonaryhypertension functional class progression as recognized by the WHO. Insome embodiments, the disclosure relates to methods of promoting orincreasing pulmonary hypertension Functional Class regression in a PcPHpatient comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the patient has FunctionalClass I, Functional Class II, Functional Class III, or Functional ClassIV pulmonary hypertension as recognized by the WHO. In some embodiments,the method relates to preventing or delaying patient progression fromFunctional Class I pulmonary hypertension to Functional Class IIpulmonary hypertension as recognized by the WHO. In some embodiments,the method relates to promoting patient regression from Functional ClassII pulmonary hypertension to Functional Class I pulmonary hypertensionas recognized by the WHO. In some embodiments, the method relates topreventing or delaying patient progression from Functional Class IIpulmonary hypertension to Functional Class III pulmonary hypertension asrecognized by the WHO. In some embodiments, the method relates topromoting patient regression from Functional Class III pulmonaryhypertension to Functional Class II pulmonary hypertension as recognizedby the WHO. In some embodiments, the method relates to promoting patientregression from Functional Class III pulmonary hypertension toFunctional Class I pulmonary hypertension as recognized by the WHO. Insome embodiments, the method relates to preventing or delaying patientprogression from Functional Class III pulmonary hypertension toFunctional Class IV pulmonary hypertension as recognized by the WHO. Insome embodiments, the method relates to promoting patient regressionfrom Functional Class IV pulmonary hypertension to Functional Class IIIpulmonary hypertension as recognized by the WHO. In some embodiments,the method relates to promoting patient regression from Functional ClassIV pulmonary hypertension to Functional Class II pulmonary hypertensionas recognized by the WHO. In some embodiments, the method relates topromoting patient regression from Functional Class IV pulmonaryhypertension to Functional Class I pulmonary hypertension as recognizedby the WHO.

The New York Heart Association (NYHA) functional classification (Table3) has been used to describe the severity of symptoms and exerciseintolerance in patients with pulmonary hypertension. The NYHA functionalclassification system provides a rapid assessment of patients'functional status in everyday clinical practice and is awell-established means of predicting prognosis. The four functionalclasses recognized by the NYHA functional classification system areshown in Table 3.

TABLE 3 New York Heart Association (NYHA) functional classification ofpulmonary hypertension based on severity of symptoms and physicalactivity Class I No limitation of physical activity. Ordinary physicalactivity does not cause undue breathlessness, fatigue, or palpitations.Class II Slight limitation of physical activity. Comfortable at rest,but ordinary physical activity results in undue breathlessness, fatigue,or palpitations. Class III Marked limitation of physical activity.Comfortable at rest, but less than ordinary physical activity results inundue breathlessness, fatigue, or palpitations. Class IV Unable to carryon any physical activity without discomfort. Symptoms at rest can bepresent If any physical activity is undertaken, discomfort is increased.

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH(e.g., treating, preventing, or reducing the progression rate and/orseverity of one or more complications of PcPH in WHO Group 2 and/orGroup 5 PH) comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the patient has FunctionalClass I, Functional Class II, Functional Class III, or Functional ClassIV pulmonary hypertension as recognized by the NYHA.

In some embodiments, the method relates to a patient that has FunctionalClass I pulmonary hypertension as recognized by the NYHA. In someembodiments, a patient with Functional Class I pulmonary hypertension asrecognized by the NYHA has no limitation of physical activity. In someembodiments, a patient with Functional Class I pulmonary hypertension asrecognized by the NYHA experiences physical activity that does not causeundue breathlessness, fatigue, and/or palpitations. In some embodiments,the method relates to a patient that has Functional Class II pulmonaryhypertension as recognized by the NYHA. In some embodiments, a patientwith Functional Class II pulmonary hypertension as recognized by theNYHA has slight limitation of physical activity. In some embodiments, apatient with Functional Class II pulmonary hypertension as recognized bythe NYHA experiences ordinary physical activity resulting in unduebreathlessness, fatigue, or palpitations. In some embodiments, themethod relates to a patient that has Functional Class III pulmonaryhypertension as recognized by the NYHA. In some embodiments, a patientwith Functional Class III pulmonary hypertension as recognized by theNYHA has marked limitation of physical activity. In some embodiments, apatient with Functional Class III pulmonary hypertension as recognizedby the NYHA experiences less than ordinary physical activity resultingin undue breathlessness, fatigue, or palpitations. In some embodiments,the method relates to a patient that has Functional Class IV pulmonaryhypertension as recognized by the NYHA. In some embodiments, a patientwith Functional Class IV pulmonary hypertension as recognized by theNYHA is unable to carry on any physical activity without discomfort. Insome embodiments, a patient with Functional Class IV pulmonaryhypertension as recognized by the NYHA experiences symptoms at rest, aswell as when any physical activity is undertaken, discomfort isincreased. In some embodiments, the method relates to patients havingFunctional Class II or Class III pulmonary hypertension as recognized bythe NYHA. In some embodiments, the method relates to patients havingFunctional Class II, Class III, or Class IV pulmonary hypertension asrecognized by the NYHA. In some embodiments, the method relates topatients having Functional Class I, Class II, Class III, or Class IVpulmonary hypertension as recognized by the NYHA. In some embodiments,the method delays clinical worsening of PcPH. In some embodiments, themethod delays clinical worsening of PcPH in accordance with the NYHA'sfunctional classification system for pulmonary hypertension.

In some embodiments, the disclosure relates to methods of preventing orreducing pulmonary hypertension Functional Class progression comprisingadministering to a patient in need thereof an effective amount of anActRII polypeptide (e.g., an amino acid sequence that is at least 90%identical to an amino acid sequence corresponding to residues 30-110 ofSEQ ID NO: 1). In some embodiments, the reduction in Functional Classprogression is a delay in Functional Class progression. In someembodiments, the method relates to preventing or decreasing pulmonaryhypertension functional class progression as recognized by the NYHA.

In some embodiments, the disclosure relates to methods of promoting orincreasing pulmonary hypertension Functional Class regression in a PcPHpatient comprising administering to a patient in need thereof aneffective amount of an ActRII polypeptide (e.g., an amino acid sequencethat is at least 90% identical to an amino acid sequence correspondingto residues 30-110 of SEQ ID NO: 1), wherein the patient has FunctionalClass I, Functional Class II, Functional Class III, or Functional ClassIV pulmonary hypertension as recognized by the NYHA. In someembodiments, the method relates to preventing or delaying patientprogression from Functional Class I pulmonary hypertension to FunctionalClass II pulmonary hypertension as recognized by the NYHA. In someembodiments, the method relates to promoting patient regression fromFunctional Class II pulmonary hypertension to Functional Class Ipulmonary hypertension as recognized by the NYHA. In some embodiments,the method relates to preventing or delaying patient progression fromFunctional Class II pulmonary hypertension to Functional Class IIIpulmonary hypertension as recognized by the NYHA. In some embodiments,the method relates to promoting patient regression from Functional ClassIII pulmonary hypertension to Functional Class II pulmonary hypertensionas recognized by the NYHA. In some embodiments, the method relates topromoting patient regression from Functional Class III pulmonaryhypertension to Functional Class I pulmonary hypertension as recognizedby the NYHA. In some embodiments, the method relates to preventing ordelaying patient progression from Functional Class III pulmonaryhypertension to Functional Class IV pulmonary hypertension as recognizedby the NYHA. In some embodiments, the method relates to promotingpatient regression from Functional Class IV pulmonary hypertension toFunctional Class III pulmonary hypertension as recognized by the NYHA.In some embodiments, the method relates to promoting patient regressionfrom Functional Class IV pulmonary hypertension to Functional Class IIpulmonary hypertension as recognized by the NYHA. In some embodiments,the method relates to promoting patient regression from Functional ClassIV pulmonary hypertension to Functional Class I pulmonary hypertensionas recognized by the NYHA.

In some embodiments, functional class regression is tested after thepatient has received 4 weeks of treatment utilizing an ActRIIpolypeptide disclosed herein. In some embodiments, functional classregression is tested after the patient has received 8 weeks of treatmentutilizing an ActRII polypeptide disclosed herein. In some embodiments,functional class regression is tested after the patient has received 12weeks of treatment utilizing an ActRII polypeptide disclosed herein. Insome embodiments, functional class regression is tested after thepatient has received 16 weeks of treatment utilizing an ActRIIpolypeptide disclosed herein. In some embodiments, functional classregression is tested after the patient has received 20 weeks oftreatment utilizing an ActRII polypeptide disclosed herein. In someembodiments, functional class regression is tested after the patient hasreceived 22 weeks of treatment utilizing an ActRII polypeptide disclosedherein. In some embodiments, functional class regression is tested afterthe patient has received 24 weeks of treatment utilizing an ActRIIpolypeptide disclosed herein. In some embodiments, functional classregression is tested after the patient has received 26 weeks oftreatment utilizing an ActRII polypeptide disclosed herein. In someembodiments, functional class regression is tested after the patient hasreceived 28 weeks of treatment utilizing an ActRII polypeptide disclosedherein. In some embodiments, functional class regression is tested afterthe patient has received 48 weeks of treatment utilizing an ActRIIpolypeptide disclosed herein.

Sustained Therapeutic Effect

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of PcPH ina sustained manner comprising administering to a patient in need thereofan effective amount of an ActRII polypeptide (e.g., an amino acidsequence that is at least 90% identical to an amino acid sequencecorresponding to residues 30-110 of SEQ ID NO: 1). In some embodiments,the sustained manner comprises a persistent therapeutic effect followingthe reduction in administration of an ActRII polypeptide describedherein. In some embodiments, the sustained manner comprises a persistenttherapeutic effect following the withdrawal of administration of anActRII polypeptide described herein. In some embodiments, the persistenttherapeutic effect relates to maintaining functional or hematologicmeasurements over time. In some embodiments, the persistent therapeuticeffect is measured as a sustained reduction in PVR. In some embodiments,the patient's PVR level does not increase for at least 1 week to atleast 12 weeks following withdrawal of an ActRII polypeptide treatmentdescribed herein. In some embodiments, the patient's PVR level does notincrease for at least 1 week following withdrawal of an ActRIIpolypeptide treatment described herein. In some embodiments, thepatient's PVR level does not increase for at least 2 weeks followingwithdrawal of an ActRII polypeptide treatment described herein. In someembodiments, the patient's PVR level does not increase for at least 3weeks following withdrawal of an ActRII polypeptide treatment describedherein. In some embodiments, the patient's PVR level does not increasefor at least 4 weeks following withdrawal of an ActRII polypeptidetreatment described herein. In some embodiments, the patient's PVR leveldoes not increase for at least 5 weeks following withdrawal of an ActRIIpolypeptide treatment described herein. In some embodiments, thepatient's PVR level does not increase for at least 6 weeks followingwithdrawal of an ActRII polypeptide treatment described herein. In someembodiments, the patient's PVR level does not increase for at least 1month to at least 6 months following withdrawal of an ActRII polypeptidetreatment described herein.

In certain aspects, the disclosure relates to methods of treating orpreventing cardiopulmonary remodeling associated with PcPH in a patient,comprising administering to a patient in need thereof an effectiveamount of an ActRII polypeptide, wherein said method slows down cardiacremodeling and/or reverses cardiac remodeling. In some embodiments, thereversal is a sustained reversal. In some embodiments, the cardiacremodeling is left cardiac remodeling. In some embodiments, the cardiacremodeling is right cardiac remodeling. In some embodiments, the cardiacremodeling is ventricle remodeling. In some embodiments, the ventricleremodeling is left ventricular remodeling. In some embodiments, theventricle remodeling is right ventricular remodeling. In someembodiments, the cardiac remodeling is ventricular dilation. In someembodiments, the method decreases interventricular septal end diastole.In some embodiments, the method decreases posterior wall end diastole.

In some embodiments, echocardiographic measurements may be used toassess the persistent therapeutic effect. In some embodiments, theechocardiographic measurements include, but are not limited to, RVfractional area change (RVFAC), sPAP, tricuspid annular systolicvelocity (TASV), and Tei index. In some embodiments, a patient treatedwith an ActRII polypeptide disclosed herein shows a persistenttherapeutic effect. In some embodiments, the persistent therapeuticeffect results in decreased intrusion of the ventral wall into the leftventricle. In some embodiments, the persistent therapeutic effectresults in an increase in right ventricular fractional area change(RVFAC).

Known Treatments for PcPH

There is no known cure for PcPH (e.g., WHO Group 2 and/or Group 5 PH);current methods of treatment focus on prolonging patient lifespan andenhancing patient quality of life. This is usually associated with goodexercise capacity, good right ventricle function, and a low mortalityrisk (e.g., bringing the patient to and/or keeping the patient in WHOFunctional Class I or Functional Class II). Current methods of treatmentof PcPH may include administration of: vasodilators such asprostacyclin, epoprostenol, and sildenafil; endothelin receptorantagonists such as bosentan; calcium channel blockers such asamlodipine, diltiazem, and nifedipine; anticoagulants such as warfarin;and diuretics. Treatment of PcPH has also been carried out using oxygentherapy, atrial septostomy, pulmonary thromboendarterectomy, and lungand/or heart transplantation. Each of these methods, however, suffersfrom one or multiple drawbacks which may include lack of effectiveness,serious side effects, low patient compliance, and high cost. In certainaspects, the method relate to treating, preventing, or reducing theprogression rate and/or severity of PcPH (e.g., treating, preventing, orreducing the progression rate and/or severity of one or morecomplications of PcPH in WHO Group 2 and/or Group 5 PH) comprisingadministering to a patient in need thereof an effective amount of anActRII polypeptide (e.g., an amino acid sequence that is at least 90%identical to an amino acid sequence corresponding to residues 30-110 ofSEQ ID NO: 1) in combination with one or more additional active agentsand/or supportive therapies for treating PcPH (e.g., vasodilators suchas prostacyclin, epoprostenol, and sildenafil; endothelin receptorantagonists such as bosentan; calcium channel blockers such asamlodipine, diltiazem, and nifedipine; anticoagulants such as warfarin;diuretics; oxygen therapy; atrial septostomy; pulmonarythromboendarterectomy: LVAD; implantable cardioverter-defibrillator(ICD); valve replacement; valve repair; and lung and/or hearttransplantation); bardoxolone methyl or a derivative thereof; oleanolicacid or derivative thereof.

Measuring Hematologic Parameters in a Patient

In certain embodiments, the present disclosure provides methods formanaging a patient that has been treated with, or is a candidate to betreated with, one or more one or more ActRII polypeptides of thedisclosure (e.g., an amino acid sequence that is at least 90% identicalto an amino acid sequence corresponding to residues 30-110 of SEQ IDNO: 1) by measuring one or more hematologic parameters in the patient.The hematologic parameters may be used to evaluate appropriate dosingfor a patient who is a candidate to be treated with one or more ActRIIpolypeptides of the present disclosure, to monitor the hematologicparameters during treatment, to evaluate whether to adjust the dosageduring treatment with one or more ActRII polypeptides of the disclosure,and/or to evaluate an appropriate maintenance dose of one or more ActRIIpolypeptides of the disclosure. If one or more of the hematologicparameters are outside the normal level, dosing with one or more ActRIIpolypeptides may be reduced, delayed or terminated.

Hematologic parameters that may be measured in accordance with themethods provided herein include, for example, red blood cell levels,blood pressure, iron stores, and other agents found in bodily fluidsthat correlate with increased red blood cell levels, using artrecognized methods. In other embodiments, hematologic parameters such aswhite blood cell levels, platelet levels, and neutrophil levels may bemeasured using art recognized methods. Such parameters may be determinedusing a blood sample from a patient. Increases in red blood cell levels,hemoglobin levels, and/or hematocrit levels may cause increases in bloodpressure. Decreases in white blood cell levels, platelet levels, and/orneutrophil levels may indicate a need to decrease, delay, or discontinuetreatment of the administration of one or more ActRII polypeptides ofthe disclosure.

In one embodiment, if one or more hematologic parameters are outside thenormal range or on the high side of normal in a patient who is acandidate to be treated with one or more ActRII polypeptides, then onsetof administration of the one or more ActRII polypeptides of thedisclosure may be delayed until the hematologic parameters have returnedto a normal or acceptable level either naturally or via therapeuticintervention. For example, if a candidate patient is hypertensive orpre-hypertensive, then the patient may be treated with a blood pressurelowering agent in order to reduce the patient's blood pressure. Anyblood pressure lowering agent appropriate for the individual patient'scondition may be used including, for example, diuretics, adrenergicinhibitors (including alpha blockers and beta blockers), vasodilators,calcium channel blockers, angiotensin-converting enzyme (ACE)inhibitors, or angiotensin II receptor blockers. Blood pressure mayalternatively be treated using a diet and exercise regimen. Similarly,if a candidate patient has iron stores that are lower than normal, or onthe low side of normal, then the patient may be treated with anappropriate regimen of diet and/or iron supplements until the patient'siron stores have returned to a normal or acceptable level. For patientshaving higher than normal red blood cell levels and/or hemoglobin levels(e.g., hemoglobin levels>16.0 g/dL or hemoglobin levels>18.0 g/dL), thenadministration of the one or more ActRII polypeptides of the disclosuremay be delayed or reduced until the levels have returned to a normal oracceptable level. In some embodiments, a normal or acceptable level ofhemoglobin includes patients with hemoglobin levels between 8-15 g/dl.In some embodiments, a normal or acceptable level of hemoglobin includespatients with hemoglobin levels of <18 g/dl. In some embodiments, anormal or acceptable level of hemoglobin increase over time includespatients whose hemoglobin levels increase less than 2 g/dL over thefirst period of time in treatment. In some embodiments, the first periodof time is 3 weeks. For patients having lower than normal white bloodcell counts (e.g., leukopenia; white blood cell count<3000/mm³ or<3.0×10⁹/L (Grade 2)), then administration of the one or more ActRIIpolypeptides of the disclosure may be delayed or reduced until thelevels have returned to a normal or acceptable level. For patientshaving lower than normal white blood cell counts (e.g., leukopenia;white blood cell count<2000/mm³ or <2.0×10⁹/L (Grade 3)), thenadministration of the one or more ActRII polypeptides of the disclosuremay be delayed or reduced until the levels have returned to a normal oracceptable level. For patients having lower than normal platelet counts(e.g., thrombocytopenia; platelet count<75,000/mm³ or <75.0×10⁹/L (Grade2)), then administration of the one or more ActRII polypeptides of thedisclosure may be delayed or reduced until the levels have returned to anormal or acceptable level. For patients having lower than normalplatelet counts (e.g., thrombocytopenia; platelet count<50,000/mm³ or<50.0×10⁹/L (Grade 3)), then administration of the one or more ActRIIpolypeptides of the disclosure may be delayed or reduced until thelevels have returned to a normal or acceptable level. For patientshaving lower than normal neutrophil counts (e.g., neutropenia;neutrophil count<1500/mm³ or <1.5×10⁹/L (Grade 2)), then administrationof the one or more ActRII polypeptides of the disclosure may be delayedor reduced until the levels have returned to a normal or acceptablelevel. For patients having lower than normal neutrophil counts (e.g.,neutropenia; neutrophil count<1000/mm³ or <1.0×10⁹/L (Grade 3)), thenadministration of the one or more ActRII polypeptides of the disclosuremay be delayed or reduced until the levels have returned to a normal oracceptable level.

In certain embodiments, if one or more hematologic parameters areoutside the normal range or on the high side of normal in a patient whois a candidate to be treated with one or more ActRII polypeptides, thenthe onset of administration may not be delayed. However, the dosageamount or frequency of dosing of the one or more ActRII polypeptidesofthe disclosure may be set at an amount that would reduce the risk ofan unacceptable increase in the hematologic parameters arising uponadministration of the one or more ActRII polypeptides of the disclosure.Alternatively, a therapeutic regimen may be developed for the patientthat combines one or more ActRII polypeptides with a therapeutic agentthat addresses the undesirable level of the hematologic parameter. Forexample, if the patient has elevated blood pressure, then a therapeuticregimen may be designed involving administration of one or more ActRIIpolypeptides and a blood pressure lowering agent. For a patient havinglower than desired iron stores, a therapeutic regimen may be developedinvolving one or more ActRII polypeptides of the disclosure and ironsupplementation.

In one embodiment, baseline parameter(s) for one or more hematologicparameters may be established for a patient who is a candidate to betreated with one or more ActRII polypeptides of the disclosure and anappropriate dosing regimen established for that patient based on thebaseline value(s). Alternatively, established baseline parameters basedon a patient's medical history could be used to inform an appropriateActRII polypeptide dosing regimen for a patient. For example, if ahealthy patient has an established baseline blood pressure reading thatis above the defined normal range it may not be necessary to bring thepatient's blood pressure into the range that is considered normal forthe general population prior to treatment with the one or more ActRIIpolypeptides of the disclosure. A patient's baseline values for one ormore hematologic parameters prior to treatment with one or more ActRIIpolypeptides of the disclosure may also be used as the relevantcomparative values for monitoring any changes to the hematologicparameters during treatment with the one or more ActRII polypeptides ofthe disclosure.

In certain embodiments, one or more hematologic parameters are measuredin patients who are being treated with one or more ActRII polypeptides.The hematologic parameters may be used to monitor the patient duringtreatment and permit adjustment or termination of the dosing with theone or more ActRII polypeptides of the disclosure or additional dosingwith another therapeutic agent. For example, if administration of one ormore ActRII polypeptides results in an increase in blood pressure, redblood cell level, or hemoglobin level, or a reduction in iron stores,white blood cell count, platelet count, or absolute neutrophil count,then the dose of the one or more ActRII polypeptides of the disclosuremay be reduced in amount or frequency in order to decrease the effectsof the one or more ActRII polypeptides of the disclosure on the one ormore hematologic parameters. If administration of one or more ActRIIpolypeptides results in a change in one or more hematologic parametersthat is adverse to the patient, then the dosing of the one or moreActRII polypeptides of the disclosure may be terminated eithertemporarily, until the hematologic parameter(s) return to an acceptablelevel, or permanently. Similarly, if one or more hematologic parametersare not brought within an acceptable range after reducing the dose orfrequency of administration of the one or more ActRII polypeptides ofthe disclosure, then the dosing may be terminated. As an alternative, orin addition to, reducing or terminating the dosing with the one or moreActRII polypeptides of the disclosure, the patient may be dosed with anadditional therapeutic agent that addresses the undesirable level in thehematologic parameter(s), such as, for example, a blood pressurelowering agent or an iron supplement. For example, if a patient beingtreated with one or more ActRII polypeptides has elevated bloodpressure, then dosing with the one or more ActRII polypeptides of thedisclosure may continue at the same level and a blood-pressure-loweringagent is added to the treatment regimen, dosing with the one or moreantagonist of the disclosure may be reduced (e.g., in amount and/orfrequency) and a blood-pressure-lowering agent is added to the treatmentregimen, or dosing with the one or more antagonist of the disclosure maybe terminated and the patient may be treated with ablood-pressure-lowering agent.

Measuring Various Parameters Over Time

In certain embodiments, one or more of the measurements of pulmonaryhypertension (e.g., PcPH) described herein can be measured over variousperiods of treatment time. In some embodiments, one or more of themeasurements of pulmonary hypertension described herein is measuredafter the patient has received 4 weeks of treatment utilizing an ActRIIpolypeptide disclosed herein. In some embodiments, one or more of themeasurements of pulmonary hypertension described herein is measuredafter the patient has received 8 weeks of treatment utilizing an ActRIIpolypeptide disclosed herein. In some embodiments, one or more of themeasurements of pulmonary hypertension described herein is measuredafter the patient has received 12 weeks of treatment utilizing an ActRIIpolypeptide disclosed herein. In some embodiments, one or more of themeasurements of pulmonary hypertension described herein is measuredafter the patient has received 16 weeks of treatment utilizing an ActRIIpolypeptide disclosed herein. In some embodiments, one or more of themeasurements of pulmonary hypertension described herein is measuredafter the patient has received 20 weeks of treatment utilizing an ActRIIpolypeptide disclosed herein. In some embodiments, one or more of themeasurements of pulmonary hypertension described herein is measuredafter the patient has received 22 weeks of treatment utilizing an ActRIIpolypeptide disclosed herein. In some embodiments, one or more of themeasurements of pulmonary hypertension described herein is measuredafter the patient has received 24 weeks of treatment utilizing an ActRIIpolypeptide disclosed herein. In some embodiments, one or more of themeasurements of pulmonary hypertension described herein is measuredafter the patient has received 26 weeks of treatment utilizing an ActRIIpolypeptide disclosed herein. In some embodiments, one or more of themeasurements of pulmonary hypertension described herein is measuredafter the patient has received 28 weeks of treatment utilizing an ActRIIpolypeptide disclosed herein. In some embodiments, one or more of themeasurements of pulmonary hypertension described herein is measuredafter the patient has received 48 weeks of treatment utilizing an ActRIIpolypeptide disclosed herein.

5. Pharmaceutical Compositions & Modes of Administration

In certain embodiments, the therapeutic methods of the disclosureinclude administering the composition systemically, or locally as animplant or device. When administered, the therapeutic composition foruse in this disclosure is in a substantially pyrogen-free, orpyrogen-free, physiologically acceptable form. Therapeutically usefulagents other than the ActRII polypeptides which may also optionally beincluded in the composition as described above, may be administeredsimultaneously or sequentially with the subject compounds in the methodsdisclosed herein.

Typically, protein therapeutic agents disclosed herein will beadministered parentally, and particularly intravenously orsubcutaneously. Pharmaceutical compositions suitable for parenteraladministration may comprise one or more ActRII polypeptides incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalcompositions of the disclosure include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants. The formulations can bepresented in unit-dose or multi-dose sealed containers, such as ampulesand vials, and can be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of a sterile liquid excipient, for example,water, for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind described herein.

The compositions and formulations may, if desired, be presented in apack or dispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

Further, the composition may be encapsulated or injected in a form fordelivery to a target tissue site. In certain embodiments, compositionsof the present invention may include a matrix capable of delivering oneor more therapeutic compounds (e.g., ActRII polypeptides) to a targettissue site, providing a structure for the developing tissue andoptimally capable of being resorbed into the body. For example, thematrix may provide slow release of the ActRII polypeptide. Such matricesmay be formed of materials presently in use for other implanted medicalapplications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalcium phosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalcium phosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

In certain embodiments, methods of the invention can be administered fororally, e.g., in the form of capsules, cachets, pills, tablets, lozenges(using a flavored basis, usually sucrose and acacia or tragacanth),powders, granules, or as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion, or as an elixir or syrup, or as pastilles (using an inertbase, such as gelatin and glycerin, or sucrose and acacia) and/or asmouth washes and the like, each containing a predetermined amount of anagent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more therapeuticcompounds of the present invention may be mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof, and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the subject compounds of the disclosure (e.g., ActRII polypeptides).The various factors include, but are not limited to, the patient's age,sex, and diet, the severity disease, time of administration, and otherclinical factors. Optionally, the dosage may vary with the type ofmatrix used in the reconstitution and the types of compounds in thecomposition.

In some embodiments, a patient's hematologic parameters can be monitoredby periodic assessments in order to determine if they have higher thannormal red blood cell levels and/or hemoglobin levels (e.g., hemoglobinlevels>16.0 g/dL or hemoglobin levels >18.0 g/dL). In some embodiments,patient's having higher than normal red blood cell levels and/orhemoglobin levels may receive a delayed or reduced dose until the levelshave returned to a normal or acceptable level.

The probability of a patient having hemoglobin levels greater than 18g/dL or increases in hemoglobin of greater than 2 g/dL may be higherduring initial treatment with an ActRII polypeptide. In certainembodiments, a dosing regimen can be used to prevent, ameliorate, ordecrease the adverse changes in hemoglobin levels. In some embodiments,ActRII polypeptides of the disclosure are administered using a dosingregimen. In some embodiments, the method comprises administering adosing regimen of a therapeutically effective amount of an ActRIIpolypeptide as disclosed herein to a patient, comprising a first dose ofbetween 0.1 mg/kg and 1.0 mg/kg of said polypeptide for a first periodof time, and a second dose of between 0.1 mg/kg and 1.0 mg/kg of saidpolypeptide subsequently administered for a second period of time. Insome embodiments, the method comprises administering a dosing regimen oftherapeutically effective amount of an ActRII polypeptide as disclosedherein to a patient, comprising a first dose of between 0.1 mg/kg and1.0 mg/kg of said polypeptide for a first period of time, a second doseof between 0.1 mg/kg and 1.0 mg/kg of said polypeptide administered fora second period of time, and a third dose of between 0.1 mg/kg and 1.0mg/kg of said polypeptide subsequently administered for a third periodof time. In some embodiments, the first dose of ActRII polypeptide isadministered to a patient in an amount from about 0.2 mg/kg to about 0.4mg/kg. In some embodiments, the first dose of ActRII polypeptide isadministered to a patient at a dose of 0.3 mg/kg. In some embodiments,the second dose of ActRII polypeptide is administered to a patient in anamount from about 0.5 mg/kg to about 0.8 mg/kg. In some embodiments, thesecond dose of ActRII polypeptide is administered to a patient at a doseof 0.7 mg/kg. In some embodiments, the third dose of ActRII polypeptideis administered to a patient in an amount from about 0.2 mg/kg to about0.4 mg/kg. In some embodiments, the third dose of ActRII polypeptide isadministered to a patient at a dose of 0.3 mg/kg.

In some embodiments, the dosing regimen comprises administering a firstdose of ActRII polypeptide to a patient in an amount of 0.3 mg/kgfollowed by administration of a second dose of ActRII polypeptide to thepatient in an amount of 0.7 mg/kg. In some embodiments, the dosingregimen comprises administering a first dose of ActRII polypeptide to apatient in an amount of 0.3 mg/kg, administering a second dose of ActRIIpolypeptide to the patient in an amount of 0.7 mg/kg, and administeringa third dose of ActRII polypeptide to the patient in an amount of 0.3mg/kg. In some embodiments, the second dose exceeds the first dose. Insome embodiments, the first dose exceeds the second dose. In someembodiments, the third dose exceeds the second dose. In someembodiments, the second dose exceeds the third dose. In someembodiments, the first period of time is at least 3 weeks. In someembodiments, the second period of time is at least 3 weeks. In someembodiments, the third period of time is at least 3 weeks. In someembodiments, the second period of time is at least 21 weeks. In someembodiments, the second period of time is at least 45 weeks. In someembodiments, the second period of time exceeds the first period of time.In some embodiments, the third period of time exceeds the first periodof time. In some embodiments, the third period of time exceeds thesecond period of time.

In some embodiments, the change in dosing between the first dose and thesecond dose is determined by the attending physician considering variousfactors (e.g., hemoglobin levels). In some embodiments, the change indosing between the second dose and the third dose is determined by theattending physician considering various factors (e.g., hemoglobinlevels). In some embodiments, the various factors include, but are notlimited to, the patient's change in hematologic parameters over a periodof time. In some embodiments, a patient's hematologic parameters aremonitored in order to determine if they have higher than normal redblood cell levels and/or hemoglobin levels (e.g., hemoglobinlevels >16.0 g/dL or hemoglobin levels >18.0 g/dL). In some embodiments,a patient's hematologic parameters are monitored in order to determineif they have a higher than normal increase in hemoglobin levels over aperiod of time (e.g., hemoglobin level increase of >2 g/dL in less than3 weeks). In some embodiments, the patient's dose of an ActRIIpolypeptide as disclosed herein will be decreased (e.g., decrease indose from 0.7 mg/kg to 0.3 mg/kg) if one or more of the patient'shematologic parameters before or during treatment is abnormal. In someembodiments, the patient's dose of an ActRII polypeptide as disclosedherein will be maintained (e.g., maintained at 0.3 mg/kg or 0.7 mg/kg)if one or more of the patient's hematologic parameters before or duringtreatment is abnormal.

In some embodiments, the dosing regimen prevents, ameliorates, ordecreases adverse effects of the ActRII polypeptide. In someembodiments, administration of an ActRII polypeptide in accordance withthe dosage regimen as provided herein results in decreased adverse sideeffects. In some embodiments, administration of an ActRII polypeptide inaccordance with the dosage regimen as provided herein decreases theprobability of having hemoglobin levels greater than 18 g/dL during thefirst period of time. In some embodiments, administration of an ActRIIpolypeptide in accordance with the dosage regimen as provided hereindecreases the probability of having hemoglobin levels greater than 18g/dL in the first 3 weeks of treatment. In some embodiments,administration of an ActRII polypeptide in accordance with the dosageregimen as provided herein decreases the probability of increasinghemoglobin levels by greater than 2 g/dL during the first period oftime. In some embodiments, administration of an ActRII polypeptide inaccordance with the dosage regimen as provided herein decreases theprobability of increasing hemoglobin levels by greater than 2 g/dL inthe first 3 weeks of treatment.

In some embodiments, ActRII polypeptides of the disclosure areadministered at a dosing range of 0.1 mg/kg to 2.0 mg/kg. In someembodiments, ActRII polypeptides of the disclosure are administered at0.1 mg/kg. In some embodiments, ActRII polypeptides of the disclosureare administered at 0.2 mg/kg. In some embodiments, ActRII polypeptidesof the disclosure are administered at 0.3 mg/kg. In some embodiments,ActRII polypeptides of the disclosure are administered at 0.4 mg/kg. Insome embodiments, ActRII polypeptides of the disclosure are administeredat 0.5 mg/kg. In some embodiments, ActRII polypeptides of the disclosureare administered at 0.6 mg/kg. In some embodiments, ActRII polypeptidesof the disclosure are administered at 0.7 mg/kg. In some embodiments,ActRII polypeptides of the disclosure are administered at 0.8 mg/kg. Insome embodiments, ActRII polypeptides of the disclosure are administeredat 0.9 mg/kg. In some embodiments, ActRII polypeptides of the disclosureare administered at 1.0 mg/kg. In some embodiments, ActRII polypeptidesof the disclosure are administered at 1.1 mg/kg. In some embodiments,ActRII polypeptides of the disclosure are administered at 1.2 mg/kg. Insome embodiments, ActRII polypeptides of the disclosure are administeredat 1.3 mg/kg. In some embodiments, ActRII polypeptides of the disclosureare administered at 1.4 mg/kg. In some embodiments, ActRII polypeptidesof the disclosure are administered at 1.5 mg/kg. In some embodiments,ActRII polypeptides of the disclosure are administered at 1.6 mg/kg. Insome embodiments, ActRII polypeptides of the disclosure are administeredat 1.7 mg/kg. In some embodiments, ActRII polypeptides of the disclosureare administered at 1.8 mg/kg. In some embodiments, ActRII polypeptidesof the disclosure are administered at 1.9 mg/kg. In some embodiments,ActRII polypeptides of the disclosure are administered at 2.0 mg/kg.

In certain embodiments, ActRII polypeptides of the disclosure areadministered once a day. In certain embodiments, ActRII polypeptides ofthe disclosure are administered twice a day. In certain embodiments,ActRII polypeptides of the disclosure are administered once a week. Incertain embodiments, ActRII polypeptides of the disclosure areadministered twice a week. In certain embodiments, ActRII polypeptidesof the disclosure are administered three times a week. In certainembodiments, ActRII polypeptides of the disclosure are administeredevery two weeks. In certain embodiments, ActRII polypeptides of thedisclosure are administered every three weeks. In certain embodiments,ActRII polypeptides of the disclosure are administered every four weeks.In certain embodiments, ActRII polypeptides of the disclosure areadministered every month.

In certain embodiments, the present invention also provides gene therapyfor the in vivo production of ActRII polypeptides. Such therapy wouldachieve its therapeutic effect by introduction of the ActRII polypeptidepolynucleotide sequences into cells or tissues having the disorders aslisted above. Delivery of ActRII polypeptide polynucleotide sequencescan be achieved using a recombinant expression vector such as a chimericvirus or a colloidal dispersion system. Preferred for therapeuticdelivery of ActRII polypeptide polynucleotide sequences is the use oftargeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. Retroviral vectors can be madetarget-specific by attaching, for example, a sugar, a glycolipid, or aprotein. Preferred targeting is accomplished by using an antibody. Thoseof skill in the art will recognize that specific polynucleotidesequences can be inserted into the retroviral genome or attached to aviral envelope to allow target specific delivery of the retroviralvector containing the ActRII polypeptide. In a preferred embodiment, thevector is targeted to bone or cartilage.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for ActRII polypeptide polynucleotidesis a colloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. RNA, DNA andintact virions can be encapsulated within the aqueous interior and bedelivered to cells in a biologically active form (see e.g., Fraley, etal., Trends Biochem. Sci., 6:77, 1981). Methods for efficient genetransfer using a liposome vehicle, are known in the art, see e.g.,Mannino, et al., Biotechniques, 6:682, 1988. The composition of theliposome is usually a combination of phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

The disclosure provides formulations that may be varied to include acidsand bases to adjust the pH; and buffering agents to keep the pH within anarrow range.

6. Kits

The present disclosure provides a kit comprising a lyophilizedpolypeptide and an injection device. In certain embodiments, thelyophilized polypeptide comprises an ActRII polypeptide (e.g., apolypeptide that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical toamino acids 30-110 of SEQ ID NO: 1), or fragments, functional variants,or modified forms thereof. In certain embodiments, the lyophilizedpolypeptide binds to one or more ligands selected from the groupconsisting of activin A, activin B, and GDF11. In certain suchembodiments, the lyophilized polypeptide further binds to one or moreligands selected from the group consisting of BMP10, GDF8, and BMP6. Incertain embodiments, the lyophilized polypeptide binds to activin and/orGDF11.

In some embodiments, the lyophilized polypeptide comprises a polypeptidethat comprises, consists essentially of, or consists of an amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to aportion of a polypeptide beginning at a residue corresponding to any oneof amino acids 21-30 (e.g., beginning at any one of amino acids 21, 22,23, 24, 25, 26, 27, 28, 29, or 30) of SEQ ID NO: 1 and ending at aposition corresponding to any one amino acids 110-135 (e.g., ending atany one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, or 135) of SEQ ID NO: 1. In certain such embodiments, thepolypeptide comprises an amino acid sequence that is least 90%, 95%, or99% identical to an amino acid sequence corresponding to residues 30-110of SEQ ID NO: 1, wherein the polypeptide binds to activin and/or GDF11.In certain embodiments, the polypeptide comprises the amino acidsequence corresponding to residues 30-110 of SEQ ID NO: 1. In otherembodiments, the polypeptide consists of the amino acid sequencecorresponding to residues 30-110 of SEQ ID NO: 1. In certainembodiments, the polypeptide is a polypeptide comprising an amino acidsequence that is at least 90%, 95%, or 99% identical to the amino acidsequence corresponding to residues 21-135 of SEQ ID NO: 1. In certainembodiments, the polypeptide comprises the amino acid sequencecorresponding to residues 21-135 of SEQ ID NO: 1. In other embodiments,the polypeptide consists of the amino acid sequence corresponding toresidues 21-135 of SEQ ID NO: 1.

In some embodiments, the lyophilized polypeptide comprises an amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 2. In certain embodiments, thepolypeptide consists essentially of the amino acid sequence of SEQ IDNO: 2. In other embodiments, the polypeptide consists of the amino acidsequence of SEQ ID NO: 2.

In some embodiments, the lyophilized polypeptide comprises an amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 3. In certain embodiments, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 3. Inother embodiments, the polypeptide consists of the amino acid sequenceof SEQ ID NO: 3.

In certain embodiments of the foregoing, the lyophilized polypeptidecomprises a fusion protein further comprising an Fc domain of animmunoglobulin. In certain such embodiments, the Fc domain of theimmunoglobulin is an Fc domain of an IgG1 immunoglobulin. In otherembodiments, the fusion protein further comprises a linker domainpositioned between the polypeptide domain and the Fc domain of theimmunoglobulin. In certain embodiments, the linker domain is selectedfrom the group consisting of: TGGG (SEQ ID NO: 20), TGGGG (SEQ ID NO:18), SGGGG (SEQ ID NO: 19), GGGGS (SEQ ID NO: 22), GGG (SEQ ID NO: 16),GGGG (SEQ ID NO: 17), and SGGG (SEQ ID NO: 21). In certain embodiments,the linker domain comprises TGGG (SEQ ID NO: 20).

In certain embodiments, the lyophilized polypeptide comprises an aminoacid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical tothe amino acid sequence of SEQ ID NO: 23. In certain embodiments, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 23. Inother embodiments, the polypeptide consists of the amino acid sequenceof SEQ ID NO: 23.

In certain embodiments, the lyophilized polypeptide comprises an aminoacid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical tothe amino acid sequence of SEQ ID NO: 30. In certain embodiments, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 30. Inother embodiments, the polypeptide consists of the amino acid sequenceof SEQ ID NO: 30.

In certain embodiments, the lyophilized polypeptide comprises an aminoacid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical tothe amino acid sequence of SEQ ID NO: 41. In certain embodiments, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 41. Inother embodiments, the polypeptide consists of the amino acid sequenceof SEQ ID NO: 41.

In certain embodiments, the lyophilized polypeptide is part of ahomodimer protein complex.

In certain embodiments, the polypeptide is glycosylated.

The present disclosure provides a kit comprising a sterile powdercomprising a lyophilized polypeptide as disclosed herein and aninjection device. In some embodiments of the kits disclosed herein, thesterile powder comprising a lyophilized polypeptide is pre-filled in oneor more containers, such as one or more vials [FIG. 21 (1)].

In certain embodiments, the pH range for the sterile powder comprising alyophilized polypeptide is from 7 to 8. In some embodiments, the sterilepowder comprising a lyophilized polypeptide further comprises abuffering agent. In some embodiments, the buffering agent may be addedin an amount of at least 10 mM. In some embodiments, the buffering agentmay be added in an amount in the range of between about 10 mM to about200 mM. In some embodiments, the buffering agent comprises citric acidmonohydrate and/or trisodium citrate dehydrate.

In some embodiments, the sterile powder comprising a lyophilizedpolypeptide further comprises a surfactant. In some embodiments, thesurfactant comprises a polysorbate. In some embodiments, the surfactantcomprises polysorbate 80.

In some embodiments, the sterile powder comprising a lyophilizedpolypeptide further comprises a lyoprotectant. In some embodiments, thelyoprotectant comprises a sugar, such as disaccharides (e.g, sucrose).In some embodiments, the lyoprotectant comprises sucrose, trehalose,mannitol, polyvinylpyrrolidone (PVP), dextrose, and/or glycine. In someembodiments, the lyoprotectant comprises sucrose. In some embodiments,the sterile powder comprises the lyoprotectant and lyophilizedpolypeptide in a weight ratio of at least 1:1 lyophilized polypeptide tolyoprotectant. In some embodiments, the sterile powder comprises thelyoprotectant and lyophilized polypeptide in a weight ratio of from 1:1to 1:10 lyophilized polypeptide to lyoprotectant. In some embodiments,the sterile powder comprises the lyoprotectant and lyophilizedpolypeptide in a weight ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, or 1:10 lyophilized polypeptide to lyoprotectant. In someembodiments, the sterile powder comprises the lyoprotectant andlyophilized polypeptide in a weight ratio of 1:6 lyophilized polypeptideto lyoprotectant. In certain embodiments of the foregoing, the sterilepowder comprises lyoprotectant in an amount sufficient to stabilize thelyophilized polypeptide.

In certain embodiments of the kits disclosed herein, the injectiondevice comprises a syringe [FIG. 21 (2)]. In certain such embodiments,the syringe is pre-filled with a reconstitution solution. In someembodiments, the reconstitution solution comprises a pharmaceuticallyacceptable carrier and/or excipient. In some embodiments, thepharmaceutically acceptable carrier comprises aqueous solutions such aswater, physiologically buffered saline, or other solvents or vehiclessuch as glycols, glycerol, oils or injectable organic esters. In someembodiments, the pharmaceutically acceptable excipient comprises apharmaceutically acceptable excipient selected from calcium phosphates,calcium carbonates, calcium sulfates, halites, metallic oxides, sugars,sugar alcohols, starch, glycols, povidones, mineral hydrocarbons,acrylic polymers, fatty alcohols, mineral stearates, glycerin, and/orlipids. In certain embodiments, the reconstitution solution comprisespharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions. In certain suchembodiments, the reconstitution solution comprises antioxidants,buffers, bacteriostats, and/or solutes which render the formulationisotonic with the blood of the intended recipient. In other embodiments,the reconstitution solution comprises suspending or thickening agents.

In certain embodiments of the kits disclosed herein, the kit furthercomprises a vial adapter [FIG. 21 (3)]. In some embodiments, the vialpre-filled with sterile powder comprising a lyophilized polypeptideattaches to one end of the vial adapter. In some embodiments, thesyringe pre-filled with a reconstitution solution as disclosed hereinattaches to an end of the vial adapter. In some embodiments, the syringepre-filled with a reconstitution solution as disclosed herein and thevial pre-filled with sterile powder comprising a lyophilized polypeptideare attached to opposite ends of the vial adapter. In some embodiments,the reconstitution solution is transferred from the pre-filled syringeto the vial. In some embodiments, transfer of the reconstitutionsolution to the vial pre-filled with sterile powder comprising alyophilized polypeptide reconstitutes the lyophilized polypeptide into asterile injectable solution. In some embodiments, the lyophilizedpolypeptide is reconstituted into a sterile injectable solution. In someembodiments, the lyophilized polypeptide is reconstituted into a sterileinjectable solution prior to use.

In other embodiments of the kits disclosed herein, the kit furthercomprises a pump apparatus. In certain embodiments, the pump apparatuscomprises an electromechanical pumping assembly. In certain embodiments,the pump apparatus comprises a reservoir for holding a sterileinjectable solution. In certain embodiments, the reservoir holds 1 mL ofsterile injectable solution. In certain embodiments, the pump apparatuscomprises one or more vials or cartridges comprising a sterileinjectable solution. In certain embodiments, the vials or cartridges areprefilled with sterile injectable solution. In certain embodiments, thevials or cartridges comprise sterile injectable solution reconstitutedfrom a lyophilized polypeptide. In certain embodiments, the reservoir iscoupled to the vial or cartridge. In certain embodiments, the vial orcartridge holds 1-20 mL of sterile injectable solution. In certainembodiments, the electromechanical pumping assembly comprises a pumpchamber. In certain embodiments, the electromechanical pumping assemblyis coupled to the reservoir. In certain embodiments, the sterileinjectable solution is received from the reservoir into the pumpchamber. In some embodiments, the electromechanical pumping assemblycomprises a plunger that is disposed such that sterile injectablesolution in the pump chamber is in direct contact with the plunger. Incertain embodiments, a sterile injectable solution is received from thereservoir into the pump chamber during a first pumping phase, and isdelivered from the pump chamber to a subject during a second pumpingphase. In certain embodiments, the electromechanical pumping assemblycomprises control circuitry. In certain embodiments, control circuitrydrives the plunger to (a) draw the sterile injectable solution into thepump chamber during the first pumping phase and (b) deliver the sterileinjectable solution from the pump chamber in a plurality of discretemotions of the plunger during the second pumping phase, therebydelivering the therapeutic substance to the subject in a plurality ofcontrolled and discrete dosages throughout the second pumping phase. Incertain embodiments, a cycle of alternating the first and second pumpingphases may be repeated until a desired dose is administered. In certainembodiments, the pump apparatus is coupled to a wearable patch. Incertain embodiments, the pump apparatus is a wearable pump apparatus. Insome embodiments, the pump apparatus administers a dose every 3 weeks.In some embodiments, the pump apparatus administers the dose viasubcutaneous injection

The present disclosure provides a kit used for reconstituting alyophilized polypeptide into a sterile injectable solution. In certainembodiments, the resulting sterile injectable solution is useful in themethods disclosed herein.

In certain embodiments of the kits disclosed herein, the kit furthercomprises an injectable device for use in administering the sterileinjectable solution parenterally [FIG. 21 (1, 2, 3, 4, and 5)]. In someembodiments, the sterile injectable solution is administered viasubcutaneous injection. In some embodiments, the sterile injectablesolution is administered via intradermal injection. In some embodiments,the sterile injectable solution is administered via intramuscularinjection. In some embodiments, the sterile injectable solution isadministered via intravenous injection. In some embodiments, the sterileinjectable solution is self-administered. In some embodiments, thesterile injectable solution comprises a therapeutically effective dose.In some embodiments, the therapeutically effective dose comprises aweight based dose. In some embodiments, the weight based dose is 0.3mg/kg. In some embodiments, the weight based dose is 0.7 mg/kg.

In some embodiments of the kits disclosed herein, the kit furthercomprises one or more vials or cartridges containing the lyophilizedpolypeptide. In some embodiments, the kit comprises at least two vialsor cartridges containing the lyophilized polypeptide. In someembodiments, the kit comprises at least three vials or cartridgescontaining the lyophilized polypeptide. In some embodiments, the twovials can contain the same or different amounts of the lyophilizedpolypeptide. In some embodiments, the vials or cartridges comprise avial or cartridge containing between 25 mg to 60 mg of lyophilizedpolypeptide. In some embodiments, at least one of the vials orcartridges comprise a vial or cartridge containing 60 mg of lyophilizedpolypeptide. In some embodiments, at least one of the vials orcartridges comprise a vial or cartridge containing 45 mg of lyophilizedpolypeptide. In some embodiments, at least one of the vials orcartridges comprise a vial or cartridge containing 30 mg of lyophilizedpolypeptide. In some embodiments, at least one of the vials orcartridges comprise a vial or cartridge containing 25 mg of lyophilizedpolypeptide. In some embodiments, a first vial or cartridge contains 45mg of lyophilized polypeptide and a second vial or cartridge contains 60mg of lyophilized polypeptide. In some embodiments, a first vial orcartridge contains 30 mg of lyophilized polypeptide and a second vial orcartridge contains 60 mg of lyophilized polypeptide. In someembodiments, a first vial or cartridge contains 30 mg of lyophilizedpolypeptide, a second vial or cartridge contains 45 mg of lyophilizedpolypeptide, and a third vial or cartridge contains 60 mg of lyophilizedpolypeptide. In some embodiments, a first vial or cartridge contains 25mg of lyophilized polypeptide, a second vial or cartridge contains 45 mgof lyophilized polypeptide, and a third vial or cartridge contains 60 mgof lyophilized polypeptide. In some embodiments, the one or more vialsor cartridges are refrigerated at 2-8° C.

7. Sequences

Human ActRIIB precursor protein sequence (SEQ ID NO: 39)MTAPWVALALLWGSLCAGSGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTLLTVLAYSLLPIGGLSLIVLLAFWMYRHRKPPYGHVDIHEDPGPPPPSPLVGLKPLQLLEIKARGRFGCVWKAQLMNDFVAVKIFPLQDKQSWQSEREIFSTPGMKHENLLQFIAAEKRGSNLEVELWLITAFHDKGSLTDYLKGNIITWNELCHVAETMSRGLSYLHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFGLAVRFEPGKPPGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELVSRCKAADGPVDEYMLPFEEEIGQHPSLEELQEVVVHKKMRPTIKDHWLKHPGLAQLCVTIEECWDHDAEARLSAGCVEERVSLIRRSVNGTTSDCLVSLVTSVTNVDLPPKESSIProcessed (mature) extracellular ActRIIB polypeptide sequence(SEQ ID NO: 31)GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT Processed (mature) extracellular ActRIIB polypeptide sequencewith the “tail” deleted (a Δ15 sequence) (SEQ ID NO: 40)GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA

8. Exemplification

The disclosure above will be more readily understood by reference to thefollowing examples, which are included merely for purposes ofillustration of certain embodiments of the present invention, and arenot intended to be limiting.

Example 1: ActRIIA-Fc Fusion Proteins

A soluble ActRII fusion protein was constructed that has theextracellular domain of human ActRIIA fused to a human or mouse Fcdomain with a minimal linker in between. The constructs are referred toas ActRIIA-hFc and ActRIIA-mFc, respectively.

ActRIIA-hFc is shown below as purified from CHO cell lines (SEQ ID NO:23):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K

An additional ActRIIA-hFc lacking the C-terminal lysine is shown belowas purified from CHO cell lines (SEQ ID NO: 41):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

The ActRIIA-hFc and ActRIIA-mFc proteins were expressed in CHO celllines. Three different leader sequences were considered:

(i) Honey bee mellitin (HBML): (SEQ ID NO: 24) MKFLVNVALVFMVVYISYIYA(ii) Tissue plasminogen activator (TPA): (SEQ ID NO: 25)MDAMKRGLCCVLLLCGAVFVSP  (iii) Native: (SEQ ID NO: 26)MGAAAKLAFAVFLISCSSGA.

The selected form employs the TPA leader and has the followingunprocessed amino acid sequence:

(SEQ ID NO: 27) MDAMKRGLCCVLLLCGAVFVSPGAAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

This polypeptide is encoded by the following nucleic acid sequence:

(SEQ ID NO: 28) ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCGGCGCCGCTATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTCTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCGGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCCACCGGTGGTGGAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGTCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGAATTC 

Both ActRIIA-hFc and ActRIIA-mFc were remarkably amenable to recombinantexpression. As shown in FIG. 4 , the protein was purified as a single,well-defined peak of protein. N-terminal sequencing revealed a singlesequence of—ILGRSETQE (SEQ ID NO: 29). Purification could be achieved bya series of column chromatography steps, including, for example, threeor more of the following, in any order: protein A chromatography, Qsepharose chromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange. TheActRIIA-hFc protein was purified to a purity of >98% as determined bysize exclusion chromatography and >95% as determined by SDS PAGE.

ActRIIA-hFc and ActRIIA-mFc showed a high affinity for ligands. GDF11 oractivin A were immobilized on a Biacore™ CM5 chip using standardamine-coupling procedure. ActRIIA-hFc and ActRIIA-mFc proteins wereloaded onto the system, and binding was measured. ActRIIA-hFc bound toactivin with a dissociation constant (K_(D)) of 5×10⁻¹² and bound toGDF11 with a K_(D) of 9.96×10⁻⁹. See FIG. 5 . Using a similar bindingassay, ActRIIA-hFc was determined to have high to moderate affinity forother TGF-beta superfamily ligands including, for example, activin B,GDF8, BMP6, and BMP10. ActRIIA-mFc behaved similarly.

The ActRIIA-hFc was very stable in pharmacokinetic studies. Rats weredosed with 1 mg/kg, 3 mg/kg, or 10 mg/kg of ActRIIA-hFc protein, andplasma levels of the protein were measured at 24, 48, 72, 144 and 168hours. In a separate study, rats were dosed at 1 mg/kg, 10 mg/kg, or 30mg/kg. In rats, ActRIIA-hFc had an 11-14 day serum half-life, andcirculating levels of the drug were quite high after two weeks (11μg/ml, 110 μg/ml, or 304 μg/ml for initial administrations of 1 mg/kg,10 mg/kg, or 30 mg/kg, respectively.) In cynomolgus monkeys, the plasmahalf-life was substantially greater than 14 days, and circulating levelsof the drug were 25 μg/ml, 304 μg/ml, or 1440 μg/ml for initialadministrations of 1 mg/kg, 10 mg/kg, or 30 mg/kg, respectively.

Example 2: Characterization of an ActRIIA-hFc Protein

ActRIIA-hFc fusion protein was expressed in stably transfected CHO-DUKXB11 cells from a pAID4 vector (SV40 or/enhancer, CMV promoter), using atissue plasminogen leader sequence of SEQ ID NO: 25. The protein,purified as described above in Example 1, had a sequence of SEQ ID NO:23. The Fe portion is a human IgG1 Fe sequence, as shown in SEQ ID NO:23. Protein analysis reveals that the ActRIIA-hFc fusion protein isformed as a homodimer with disulfide bonding.

The CHO-cell-expressed material has a higher affinity for activin Bligand than that reported for an ActRIIA-hFc fusion protein expressed inhuman 293 cells [see, del Re et al. (2004) J Biol Chem.279(51):53126-53135]. Additionally, the use of the TPA leader sequenceprovided greater production than other leader sequences and, unlikeActRIIA-Fc expressed with a native leader, provided a highly pureN-terminal sequence. Use of the native leader sequence resulted in twomajor species of ActRIIA-Fc, each having a different N-terminalsequence.

Example 3: Alternative ActRIIA-Fc Proteins

A variety of ActRIIA variants that may be used according to the methodsdescribed herein are described in the International Patent Applicationpublished as WO2006/012627 (see e.g., pp. 55-58), incorporated herein byreference in its entirety. An alternative construct may have a deletionof the C-terminal tail (the final 15 amino acids of the extracellulardomain of ActRIIA). The sequence for such a construct is presented below(Fc portion underlined) (SEQ ID NO: 30):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Example 4: Effects of an ActRIIA-mFc on Group 2 Pulmonary Hypertensionin a Transverse Aortic Constriction (TAC) Induced PH Mouse Model

The effects of an ActRIIA-mFc fusion protein (ActRIIA-mFc homodimer asdescribed in Example 1) was examined in a mouse model of leftventricular systolic dysfunction (also referred to as HErEF) ofpulmonary hypertension (PH). In this model, C57BL/6 mice underwenttransverse aortic constriction (TAC) to induce left heart failure, andright heart and pulmonary remodeling. See, e.g., Xiong P Y, et al.Hypertension 2018, 71(1):34-55 and Chen Y, et al. Hypertension 2012,59(6):1170-1178.

Twenty-six C57/B6 male mice (10 wks old) underwent TAC surgery and tenage-matched animals underwent a mock surgical procedure (Sham) at day 0.Two weeks after the surgery, TAC-PH mice were randomized into twogroups. i) fourteen mice were injected subcutaneously with vehiclecontrol (phosphate buffered saline (PBS)), twice weekly for 4 weeksstarting from day 14 after surgery, “TAC-PH/PBS”; and a ii) twelve micewere injected subcutaneously with ActRIIA-mFc at a dose of 10 mg/kgtwice weekly for 4 weeks starting from day 14 after TAC surgery,“TAC-PH/ActRII-mFc”. At the end of the study, echocardiography andpressure-volume catheter were performed to measure left and rightventricular remodeling and functional changes before animals wereeuthanized for heart and lung collection. Hearts and lungs of each mousewere weighed, fixed in 10% formalin, embedded in paraffin, and sectionedfor Masson's trichrome stain to assess fibrosis.

Prior to euthanasia, in vivo cardiac function was assessed bytransthoracic echocardiography (Acuson P300, 18 MHz transducer; Siemens)in conscious mice. From left ventricle (LV) short axis view, M-modeechocardiogram was acquired to measure left ventricle end diastolicdiameter (LVEDD), and left ventricle end systolic diameter (LVESD).Fractional shortening (FS) was calculated from the end-diastolicdiameter (EDD) and end-systolic diameter (ESD) using the followingequation: FS=100%×[(EDD−ESD)/EDD]. Early diastolic filling peak velocity(E), early diastolic mitral annular velocity (E′), and isovolumetricrelaxation time (IVRT) were measured from the medial or septal wall atthe mitral valve level from tissue Doppler image. LV diastolic functionwas assessed by measuring the E/E′ ratio and IVRT. Three to five beatswere averaged for each mouse measurement. Tricuspid annular planesystolic excursion (TAPSE), a parameter of global right ventricularfunction, was also measured.

On day 42, mice were anesthetized by an intraperitoneal injection ofketamine/xylazine (100/5 mg/kg) to evaluate left and right ventricularfunction by Millar pressure-volume conductance catheter. The respirationwas supported by a small animal ventilator. Thoracotomy was made through4-5 intercostal space, and the heart was exposed. A pressure-volumecatheter (1.0-Fr, PVR-1035, Millar Instruments, Houston, Tex., USA) wasinserted into the left ventricle and right ventricle from the apex.Ventricular pressure and volume were calculated with LabChart 7software. Stroke work, ejection fraction, maximum and minimum rate ofpressure development (+dp/dtm, −dp/dtm) were derived.

Compared to Sham control animals, TAC-PH mice in the PBS treatment group(TAC-PH/PBS) on day 42 were observed to have increased heart weight(HW/BW) (FIG. 10 ), reduced FS (FIG. 11 ), reduced LV ejection fraction(FIG. 12 ), increased E/E′ ratio (FIG. 13 ), and increased IVRT (FIG. 14), indicating cardiac hypertrophy and left heart failure. TAC mice alsoincreased right ventricle free wall thickness (RVFWT) (FIG. 15 ),decreased TAPSE (FIG. 16 ), increased right ventricle (RV) stroke work(FIG. 17 ), and increased minimum rate of pressure development in RV(−dp/dT_(min)) (FIG. 18 ) compared to Sham control mice, suggesting theRV remodeling and RV dysfunction. In addition, increased lung weight(LW/TL) (FIG. 19 ) and lung fibrosis (FIG. 20 ) were observed inTAC-PH/PBS mice, indicating lung remodeling caused by TAC-induced leftheart failure.

As shown in FIGS. 10-20 , ActRIIA-mFc treatment (TAC-PH/ActRIIA-mFc)relative to PBS treatment (TAC-PH/PBS) on day 42 significantly reducedcardiac hypertrophy (FIG. 10 ), elevated FS (FIG. 11 ), restored LVejection fraction (FIG. 12 ), reduced E/E′ ratio (FIG. 13 ), and reducedIVRT (FIG. 14 ). ActRIIA-mFc treatment (TAC-PH/ActRIIA-mFc) relative toPBS treatment (TAC-PH/PBS) on day 42 also significantly reduced elevatedRVFWT (FIG. 15 ), increased reduced TAPSE (FIG. 16 ), reduced elevatedRV stroke work (FIG. 17 ), and decreased increased RV −dp/dT_(min) (FIG.18 ). ActRIIA-mFc treatment (TAC-PH/ActRII-mFc) relative to PBStreatment (TAC-PH/PBS) on day 42 decreased lung weight (FIG. 19 ) andsignificantly reduced lung fibrosis (FIG. 20 ).

Together, these data demonstrate that ActRIIA-mFc is effective inameliorating various complications of Group 2 PH in a left heartfailure-induced PH model (TAC-PH). In particular, ActRIIA-mFc had asignificant effect in reducing cardiac hypertrophy, improving cardiacfunction, improving right heart remodeling and function, and reducingpulmonary remodeling and fibrosis.

Example 5: Effects of an ActRIIA-mFc on Group 2 Pulmonary Hypertensionin an HFpEF Induced PH Rat Model

The effects of an ActRIIA-mFc fusion protein (ActRIIA-mFc homodimer asdescribed in Example 1) was examined in a rat model of left ventriculardiastolic dysfunction (also referred to as HEpEF) group 2 (subgroup 2.2)pulmonary hypertension (PH). In this model, ZSF1-Lepr^(fa) Lepr^(cp)/Crlrats were challenged with semaxanib to induce HFpEF-PH (1).

Forty ZSF1 Lepr^(fa)Lepr^(cp)/Crl male mice (8wks old) and five leanrats were subcutaneously administered with a single dose of semaxanib(100 mg/kg) at day 0, and five lean rats were included as normalcontrol. Six weeks after semaxanib (SU5416) treatment, Thirty-six ZSF1Lepr^(fa)Lepr^(cp)/Crl rats were randomized into four groups: i) ninerats were injected subcutaneously with vehicle control (phosphatebuffered saline (PBS)), twice weekly for 8 weeks starting from day 42after semaxanib treatment, “ZSF1-SU/PBS”; a ii) ten rats were injectedsubcutaneously with ActRIIA-mFc at a dose of 1 mg/kg twice weekly for 8weeks starting from day 42 after semaxanib treatment,“ZSF1-SU/ActRIIA-mFc 1 mpk”; a iii) nine rats were injectedsubcutaneously with ActRIIA-mFc at a dose of 3 mg/kg twice weekly for 8weeks starting from day 42 after semaxanib treatment,“ZSF1-SU/ActRIIA-mFc 3 mpk”; and a iv) eight rats were injectedsubcutaneously with ActRIIA-mFc at a dose of 10 mg/kg twice weekly for 8weeks starting from day 42 after semaxanib treatment,“ZSF1-SU/ActRIIA-mFc 10 mpk”. At the end of the study, echocardiographyand pressure-volume catheter were performed to measure left and rightventricular remodeling and functional changes before animals wereeuthanized for heart and lung collection. Hearts and lungs of each ratwere weighed, fixed in 10% formalin, embedded in paraffin, and sectionedfor Masson's trichrome stain to assess fibrosis. Serum and urine sampleswere collected at the end of the study.

Rats were fasted overnight to measure fasting blood glucose levels atweek 14 (before treatments started), week 18 (4 weeks after treatments),and week 22, and oral glucose tolerance test was performed at week 22.Blood glucose levels were measured with a glucometer after bleeding tailvein with a 27G needle. To prepare oral glucose tolerance test, 40%glucose stock solution run through a filter to sterilize it. Afterfasting overnight, rat body weight was measured. Blood glucose level wasdetected. Then 40% glucose solution was administered via oral gavageaccording to body weight (2 g/kg). Blood glucose levels were measured at30, 60, 90, 120 minutes.

Prior to euthanization, in vivo cardiac function was assessed bytransthoracic echocardiography (Acuson P300, 18 MHz linear transducer;Siemens) in lightly anesthetized rats as described (2). From leftventricle short axis view, M-mode echocardiogram was acquired to measureinterventricular septal thickness at end diastole (IVSd), leftventricular posterior wall thickness at end diastole (LVPWd), leftventricular end diastolic diameter (LVEDD), and left ventricular endsystolic diameter (LVESD). Left ventricular mass (LVM) was assessed bythe equation: 1.05 [(LVEDD+LVPTD+IVSd)³-LVEDD³]. Early diastolic fillingpeak velocity (E), early diastolic mitral annular velocity (E′), andisovolumetric relaxation time (IVRT) were measured from the medial orseptal wall at the mitral valve level from tissue Doppler image. LVdiastolic function was assessed by measuring the E/E′ ratio and IVRT.Pulmonary arterial acceleration time (PAAT), a parameter of rightventricular function, was also measured.

Fourteen weeks after semaxanib treatment, rats were anesthetized withketamine (100 mg/kg) and xylazine (5 mg/kg) at the end of the experimentto evaluate cardiac and pulmonary hemodynamics. The respiration wassupported by a small animal ventilator. Thoracotomy was made through 4-5intercostal space, and the heart was exposed. A pressure-volume catheter(2.0-Fr, SPR-869, Millar Instruments, Houston, Tex., USA) was beinserted into the left ventricle and right ventricle from the apex.Ventricular pressure and volume were calculated with LabChart 7software. Stroke work, ejection fraction, and cardiac output werederived. After finishing left ventricular measurements, the catheter wasadvanced to the aorta, arterial blood systolic and diastolic pressurewas detected. Then the catheter returned to the left ventricle andchanged the direction laterally to enter the left atrium. Similarly,right atrial pressure was measured by moving the catheter from the rightventricle into atrium. To measure pulmonary arterial pressure, thesternum was cross-sectioned at the second inter-rib space. The rightventricular outflow tract was exposed. A hole was made with 27G needle,and then the catheter was inserted into the right ventricular outflowtract and advanced into the pulmonary artery.

Compared to lean control animals, ZSF1-SU rats in the PBS treatmentgroup (ZSF1-SU/PBS) 14-weeks after semaxanib treatment were observed tohave increased heart weight (HW/TL) (FIG. 26 ), increased IVSd (FIG. 27), and increased LVM (FIG. 28 ), preserved LV ejection fraction (FIG. 23), increased E/E′ ratio (FIG. 24 ), increased IVRT (FIG. 25 ),indicating cardiac hypertrophy and left ventricular diastolicdysfunction. ZSF1 rats also increased right ventricle free wallthickness (RVFWT) (FIG. 29 ), decreased PAAT (FIG. 30 ), and increasedRVSP (FIG. 31 ), compared to lean control rats, suggesting the pulmonaryhypertension and RV remodeling. In addition, increased fibrosis in LV,RV and lung (FIGS. 32-34 ) was observed in ZSF1-SU/PBS rats.

As shown in FIGS. 26 to 28 , ActRIIA-mFc treatment (ZSF1-SU/ActRIIA-mFc)relative to PBS treatment (ZSF1-SU/PBS) both at 3 mpk and 10 mpksignificantly reduced left heart remodeling (FIGS. 26-28 ), and reducedE/E′ ratio (FIG. 24 ), and decreased IVRT (FIG. 25 ). ActRIIA-mFctreatment (ZSF1-SU/ActRIIA-mFc) relative to PBS treatment (ZSF1-SU/PBS)both at 3 mpk and 10 mpk also significantly reduced elevated RVFWT (FIG.29 ), reduced PAAT (FIG. 30 ), and reduced elevated RVSP (FIG. 31 ).ActRIIA-mFc treatment (ZSF1-SU/ActRIIA-mFc) relative to PBS treatment(ZSF1-SU/PBS) also significantly reduced the increased fibrosis in LV,RV and lung (FIGS. 32-34 ).

In addition, compared to lean control animals, ZSF1-SU rats in the PBStreatment group (ZSF1-SU/PBS) had elevated fasting blood glucose leveland increased glucose level in urine, accompanied by glucoseintolerance. ActRIIA-mFc treatment (ZSF1-SU/ActRIIA-mFc) relative to PBStreatment (ZSF1-SU/PBS) at 1 mpk, 3 mpk and 10 mpk significantly reducedfasting blood glucose, decreased glucose level in urine, and improvedglucose tolerance (FIGS. 36-38 ).

Together, these data demonstrate that ActRIIA-mFc is effective inameliorating various complications of Group 2 PH in a left heartfailure-induced PH model (HFpEF-PH). In particular, ActRIIA-mFc had asignificant effect in reducing cardiac hypertrophy, improving diastolicfunction, improving right heart remodeling and function, decreasingpulmonary hypertension, and reducing cardiac and pulmonary remodelingand fibrosis. Furthermore, ActRIIA-mFc had a robust effect in reducingglucose levels and improving glucose tolerance. The data indicate thatother ActRII antagonists, particularly ones having activities similar toActRIIA-mFc, may be useful in the treatment of Group 2 PH, particularlyin preventing or reducing the severity various complications of Group 2PH.

Example 6: Effects of an ActRIIA-hFc Polypeptide in Patients with Cpc-PHDue to HFpEF

The effects of an ActRIIA-hFc fusion protein (SEQ ID NO: 23 as describedin Example 1) are examined in a double-blind, randomized,placebo-controlled study to evaluate the effects of the ActRIIA-hFcfusion protein versus placebo for the treatment of combined pre- andpostcapillary pulmonary hypertension (Cpc-PH) due to heart failure withpreserved ejection fraction (HFpEF).

Patients and Trial Design

Eligible patients will have confirmed Cpc-PH due to HFpEF, FunctionalClass II or III as assessed by the NYHA. Additionally, eligible patientsare between 18 to 85 years of age and have a six minute walk distancegreater than 100 meters repeated twice during screening and both valueswithin 15% of each other, calculated from the highest value. Patientsmay be receiving stable medications for heart failure or any underlyingcondition for at least 30 days before and throughout the study. Aplanned interim analysis will occur when approximately 15 participantsin each of the three treatment groups have completed 24 weeks on thestudy. Sensitivity analysis will be performed to account for anydifferences in background therapy. All patients will provide informedconsent.

Initially, approximately 90 eligible patients will be randomly assignedin a 1:1:1 ratio to one of three treatment groups: (1) placebo; (2)ActRIIA-hFc fusion protein 0.3 mg/kg; or (3) ActRIIA-hFc fusion protein0.3 mg/kg then escalating to 0.7 mg/kg. ActRIIA-hFc fusion protein orplacebo (saline) will be given by subcutaneous injection every 21 daysfor a total of 24 weeks. Safety and efficacy will be assessed atscreening and every 3 weeks for 24 weeks. See, e.g., Table 4 below.Adverse events are recorded from screening until the end of primarytreatment study visit, 8 weeks after the last dose of study drug. Aninterim analysis will occur when approximately 15 participants in eachof the 3 treatment groups have completed 24 weeks of treatment in theplacebo-controlled treatment period.

Participants who have not discontinued early from the placebo-controlledtreatment period and have had the 24-week PVR assessment will continueinto the 18-month extension period and will be treated as follows:Placebo participants will be re-randomized in a 1:1 ratio to one of thetwo ActRIIA-hFc fusion protein treatment groups utilized in theplacebo-controlled treatment period to receive either (1) ActRIIA-hFcfusion protein SC at a dose level of 0.3 mg/kg every 21 days for up to18 months in the Extension Period or (2) ActRIIA-hFc fusion protein SCat a starting dose of 0.3 mg/kg plus background therapy, then escalateto 0.7 mg/kg at Visit 12 and every 21 days for up to 18 months in theExtension Period.

TABLE 4 Efficacy Endpoints Type End points Primary end Change inpulmonary vascular resistance from baseline to 24 weeks point Key Changein 6-minute walk distance from baseline to 24 weeks secondary end pointOther • Clinical Worsening secondary  ○ Number of Clinical Worseningevents, defined as follows, at 24 end points and 48 weeks:  - Theoccurrence of any 1 of the following clinical worsening  events:hospitalization due to a cardiopulmonary indication  (a non-electivehospitalization lasting at least 24 hours in  duration caused byclinical conditions directly related to PH  and/or heart failure),administration of IV diuretics, death (all  causes), decrease in 6MWD >15% from Baseline (or the  subject was too ill to walk, and the causewas directly related  to the disease under study) at 2 consecutivevisits on different  days (except Week 24)  ○ Number of Participantswith first Clinical Worsening event, defined as above, at 24 and 48weeks  ○ Time to Clinical Worsening, defined as above • Change indyspnea score (assessed by Borg CR10 scale ®) at Week 24 from baseline •Change in hemodynamic and ECHO parameters, including but not limited tomPAP, PCWP, TAPSE, RVFAC, and LVEF at 24 weeks from baseline • Change inNT-proBNP at 24 weeks from baseline • Change in NYHA FC at 24 weeks frombaseline • Change in 6MWD at 48 weeks from baseline • Change inhemodynamic and ECHO parameters, including but not limited to PVR, mPAP,PCWP, TAPSE, RVFAC, and LVEF, at 48 weeks from baseline • Change inNT-proBNP at 48 weeks from baseline • Change in NYHA FC at 48 weeks frombaseline • Change in PVR, 6MWD and NYHA FC at week 48 from baseline inthe extension in the Placebo-Crossed treatment group • Change in PVR,6MWD and NYHA FC from week 24 to week 48 in the extension in thePlacebo-Crossed treatment group Exploratory Placebo-Controlled Treatmentand Extension Periods end points • Changes in Kansas City CardiomyopathyQuestionnaire (KCCQ) and EQ-5D-5L scores • Change from baseline indisease-related biomarkers at 24 weeks and 48 weeks • Correlation ofclinical efficacy vs. genetic phenotype

Example 7: Effects of an ActRIIA-mFc on Group 2 Pulmonary Hypertensionin a Transverse Aortic Constriction (TAC) Induced PH Mouse Model

The effects of an ActRIIA-mFc fusion protein (ActRIIA-mFc homodimer asdescribed in Example 1) was examined in a mouse model of leftventricular systolic dysfunction (also referred to as HErEF) ofpulmonary hypertension (PH) and valvular heart disease. In this model,BALB/cJ mice underwent transverse aortic constriction (TAC) to induceleft heart failure, and right heart and pulmonary remodeling. See, e.g.,Xiong P Y, et al. Hypertension 2018, 71(1):34-55 and Chen Y, et al.Hypertension 2012, 59(6):1170-1178.

Forty-four BALB/cJ male mice (10 wks old) underwent TAC surgery andfourteen age-matched animals underwent a mock surgical procedure (Sham)at day 0. Two weeks after the surgery, TAC-PH mice were randomized intothree groups. i) fourteen mice were injected subcutaneously with vehiclecontrol (phosphate buffered saline (PBS)), twice weekly for 4 weeksstarting from day 14 after surgery, “TAC PBS”; ii) fifteen mice wereinjected subcutaneously with ActRIIA-mFc at a dose of 3 mg/kg twiceweekly for 4 weeks starting from day 14 after TAC surgery, “TACActRIIA-mFc 3 mpk”; and iii) fifteen mice were injected subcutaneouslywith ActRIIA-mFc at a dose of 10 mg/kg twice weekly for 4 weeks startingfrom day 14 after TAC surgery, “TAC ActRIIA-mFc 10 mpk.” At the end ofthe study, echocardiography and pressure-volume catheter were performedto measure left and right ventricular remodeling and functional changesbefore animals were euthanized for heart and lung collection. Hearts andlungs of each mouse were weighed, fixed in 10% formalin, embedded inparaffin, and sectioned for Masson's trichrome stain to assess fibrosis.

Prior to euthanasia, in vivo cardiac function was assessed bytransthoracic echocardiography (Acuson P300, 18 MHz transducer; Siemens)in conscious mice. From left ventricle (LV) short axis view, M-modeechocardiogram was acquired to measure left ventricle end diastolicdiameter (LVEDD), and left ventricle end systolic diameter (LVESD).Fractional shortening (FS) was calculated from the end-diastolicdiameter (EDD) and end-systolic diameter (ESD) using the followingequation: FS=100%×[(EDD−ESD)/EDD]. Early diastolic filling peak velocity(E), early diastolic mitral annular velocity (E′), and isovolumetricrelaxation time (IVRT) were measured from the medial or septal wall atthe mitral valve level from tissue Doppler image. LV diastolic functionwas assessed by measuring the E/E′ ratio and IVRT. Three to five beatswere averaged for each mouse measurement. RV free wall thickness (RVFWT)was measured using M-mode in a modified parasternal long-axis viewthrough the aortic valve. Pulmonary artery acceleration time (PAAT) wasmeasured as the time from start to peak velocity of blood flow in thelumen of the main pulmonary artery distal to the pulmonary valve asobtained from the pulse-wave doppler recording.

On day 42, mice were anesthetized by an intraperitoneal injection ofketamine/xylazine (100/5 mg/kg) to evaluate left and right ventricularfunction by Millar pressure-volume conductance catheter. The respirationwas supported by a small animal ventilator. Thoracotomy was made through4-5 intercostal space, and the heart was exposed. A pressure-volumecatheter (1.0-Fr, PVR-1035, Millar Instruments, Houston, Tex., USA) wasinserted into the left ventricle and right ventricle from the apex.Ventricular pressure and volume were calculated with LabChart 7software. Ejection fraction was derived. Afterwards, animals wereeuthanized for heart and lung collection. Cardiac hypertrophy wasmeasured by heart weight (HW) normalized by tibial length (TL). Leftventricle (LV), right ventricle (RV) and lung of each mouse wereseparated, fixed in 10% formalin, embedded in paraffin, and sectionedfor Masson's trichrome stain to assess fibrosis. Serum and urine sampleswere collected at the end of the study.

Compared to Sham control animals, TAC-PH mice in the PBS treatment group(TAC PBS) on day 42 were observed to have decreased left ventricleejection fraction (FIG. 40 ), increased heart weight (HW/TL) (FIG. 41 ),increased E/E′ ratio (FIG. 42 ), increased isovolumic relaxation time(IVRT) (FIG. 43 ), and increased left ventricle fibrosis (FIG. 47 ),indicating cardiac hypertrophy and left heart failure. TAC mice also hadincreased right ventricle free wall thickness (RVFWT) (FIG. 45 ),decreased PAAT (FIG. 46 ), and increased right ventricle fibrosis (FIG.48 ) compared to Sham control mice, suggesting the RV remodeling and RVdysfunction. In addition, increased RVSP (FIG. 44 ) and increased lungfibrosis (FIG. 49 ) were observed in TAC-PH/PBS mice, indicatingpulmonary hypertension and lung remodeling caused by TAC-induced leftheart failure.

As shown in FIGS. 40-49 , ActRIIA-mFc treatment (TAC ActRIIA-mFc 3 mpkor TAC ActRIIA-mFc 10 mpk) relative to PBS treatment (TAC PBS) on day 42significantly reduced cardiac hypertrophy (FIG. 41 ), restored LVejection fraction (FIG. 40 ), reduced E/E′ ratio at 3 mpk (FIG. 42 ),and reduced IVRT (FIG. 43 ). ActRIIA-mFc treatment (TAC ActRIIA-mFc 3mpk or TAC ActRIIA-mFc 10 mpk) relative to PBS treatment (TAC PBS) onday 42 also significantly reduced elevated RVFWT (FIG. 45 ), reducedRVSP (FIG. 44 ), (FIG. 45 ), and increased PAAT (FIG. 46 ). ActRIIA-mFctreatment (TAC ActRIIA-mFc 3 mpk or TAC ActRIIA-mFc 10 mpk) relative toPBS treatment (TAC PBS) on day 42 significantly reduced lung fibrosis(FIG. 49 ), LV fibrosis (FIG. 47 ), and RV fibrosis (FIG. 48 ).

Together, these data demonstrate that ActRIIA-mFc is effective inameliorating various complications of Group 2 PH in a left heartfailure-induced PH model (TAC PH). In particular, ActRIIA-mFc had asignificant effect in reducing cardiac hypertrophy, improving cardiacfunction, improving right heart remodeling and function, improving LVfunction, and reducing pulmonary remodeling and fibrosis.

Example 8: Effects of an ActRIIA-mFc on Group 2 Pulmonary Hypertensionin an HFpEF Induced PH Rat Model

The effects of an ActRIIA-mFc fusion protein (ActRIIA-mFc homodimer asdescribed in Example 1) was examined in a rat model of left ventriculardiastolic dysfunction (also referred to as HFpEF) group 2 (subgroup 2.2)pulmonary hypertension (PH). In this model, ZSF1-Lepr^(fa)Lepr^(cp)/Crlrats were challenged with semaxanib (SU5416) to induce HFpEF-PH.

Twenty ZSF1 Lepr^(fa)Lepr^(cp)/Crl male mice (8wks old) and ten leanrats were subcutaneously administered with a single dose of semaxanib(100 mg/kg) at day 0, and ten lean rats were included as normal control.Eight weeks after semaxanib (SU5416) treatment, twenty ZSF1Lepr^(fa)Lepr^(cp)/Crl rats were randomized into two groups: i) ten ratswere injected subcutaneously with vehicle control (phosphate bufferedsaline (PBS)), twice weekly for 8 weeks starting from day 64 aftersemaxanib treatment, “Obese ZSF1 Veh”; and ii) ten rats were injectedsubcutaneously with ActRIIA-mFc at a dose of 5 mpk twice weekly for 8weeks starting from day 64 after semaxanib treatment, “Obese ZSF1ActRIIA-mFc.” At the end of the study, echocardiography andpressure-volume catheter were performed to measure left and rightventricular remodeling and functional changes before animals wereeuthanized for heart and lung collection.

Prior to euthanization, in vivo cardiac function was assessed bytransthoracic echocardiography (Acuson P300, 18 MHz linear transducer;Siemens) in lightly anesthetized rats as described. Echocardiographicassessments were conducted at week 8 (before therapy with ActRIIA-mFc orvehicle) and week 15 (after therapy) in each rat. From left ventricleshort axis view, M-mode echocardiogram was acquired to measureinterventricular septal thickness at end diastole (IVSd), leftventricular posterior wall thickness at end diastole (LVPWd), leftventricular end diastolic diameter (LVEDD), and left ventricular endsystolic diameter (LVESD). Left ventricular mass (LVM) was assessed bythe equation: 1.05 [(LVEDD+LVPTD+IVSd)³-LVEDD³]. Early diastolic fillingpeak velocity (E), early diastolic mitral annular velocity (E′), andisovolumetric relaxation time (IVRT) were measured from the medial orseptal wall at the mitral valve level from tissue Doppler image. LVdiastolic function was assessed by measuring the E/E′ ratio and IVRT.Pulmonary arterial acceleration time (PAAT), a parameter of rightventricular function, was measured. Tricuspid annular plane systolicexcursion (TAPSE), a parameter of global right ventricular function, wasalso measured. RV free wall thickness (RVFWT) was measured using M-modein a modified parasternal long-axis view through the aortic valve.Pulmonary artery acceleration time (PAAT) was measured as the time fromstart to peak velocity of blood flow in the lumen of the main pulmonaryartery distal to the pulmonary valve as obtained from the pulse-wavedoppler recording.

Sixteen weeks after semaxanib treatment, rats were anesthetized withketamine (100 mg/kg) and xylazine (5 mg/kg) at the end of the experimentto evaluate cardiac and pulmonary hemodynamics. The respiration wassupported by a small animal ventilator. Thoracotomy was made through 4-5intercostal space, and the heart was exposed. A pressure-volume catheter(2.0-Fr, SPR-869, Millar Instruments, Houston, Tex., USA) was insertedinto the left ventricle and right ventricle from the apex. Ventricularpressure and volume were calculated with LabChart 7 software. Strokework, ejection fraction, and cardiac output were derived. Afterfinishing left ventricular measurements, the catheter was advanced tothe aorta, arterial blood systolic and diastolic pressure was detected.Then the catheter returned to the left ventricle and changed thedirection laterally to enter the left atrium. Similarly, right atrialpressure was measured by moving the catheter from the right ventricleinto atrium. To measure pulmonary arterial pressure, the sternum wascross-sectioned at the second inter-rib space. The right ventricularoutflow tract was exposed. A hole was made with 27G needle, and then thecatheter was inserted into the right ventricular outflow tract andadvanced into the pulmonary artery.

Compared to lean control animals, Obese ZSF1-SU rats in the PBStreatment group (Obese ZSF1 SU/Veh) 16-weeks after semaxanib treatmentwere observed to have decreased pulmonary artery acceleration time(PAAT) (FIG. 51 ), increased RVSP (FIG. 52 ), increased right ventriclefree wall thickness (RVFWT) (FIG. 53 ), decreased tricuspid annularplane systolic excursion (TAPSE) (FIG. 54 ), and increased Fulton Index,calculated as the ratio of right ventricular weight (RV) to weight ofthe combined left ventricle and septum (LV+S) (FIG. 55 ).

As shown in FIGS. 51 and 52 , ActRIIA-mFc treatment (ObeseZSF1-SU/ActRIIA-mFc) relative to PBS treatment (Obese ZSF1-SU/Veh) at 5mpk normalized cardiopulmonary function as shown by the significantlyincreased PAAT (FIG. 51 ) and significantly reduced right ventricularsystolic pressure (RVSP) (FIG. 52 ). ActRIIA-mFc treatment (ObeseZSF1-SU/ActRIIA-mFc) relative to PBS treatment (Obese ZSF1-SU/Veh) at 5mpk also normalized right ventricular structure and function as shown bythe significantly reduced elevated RVWT (FIG. 53 ), increased TAPSE(FIG. 54 ), and the decreased Fulton index (FIG. 55 ).

Together, these data demonstrate that ActRIIA-mFc is effective inameliorating various complications of Group 2 PH in a left heartfailure-induced PH model (HFpEF-PH). In particular, ActRIIA-mFc had asignificant effect in normalizing cardiopulmonary function and innormalizing right ventricular structure and function.

We claim:
 1. A method of treating post-capillary pulmonary hypertension(PcPH), comprising administering to a patient in need thereof aneffective amount of a polypeptide comprising an amino acid sequence thatis at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acidsequence that begins at any one of amino acids 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 of SEQ ID NO: 1 and ends at any one of amino acids110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135 of SEQ IDNO:
 1. 2. A method of treating, preventing, or reducing the progressionrate and/or severity of one or more complications of post-capillarypulmonary hypertension, comprising administering to a patient in needthereof an effective amount of a polypeptide comprising an amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to anamino acid sequence that begins at any one of amino acids 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 of SEQ ID NO: 1 and ends at any one ofamino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135of SEQ ID NO:
 1. 3. The method of claim 2, wherein the one or morecomplications of post-capillary pulmonary hypertension is selected fromthe group consisting of: smooth muscle and/or endothelial cellproliferation in the pulmonary artery, angiogenesis in the pulmonaryartery, dyspnea, chest pain, pulmonary vascular remodeling, rightventricular hypertrophy, left ventricular hypertrophy, left atriumdilation, left ventricular fibrosis, right ventricular fibrosis, andpulmonary fibrosis.
 4. The method of any one of claims 1-3, wherein thePcPH is isolated post-capillary pulmonary hypertension (IpcPH).
 5. Themethod of any one of claims 1-3, wherein the PcPH is combined post- andpre-capillary PH (CpcPH).
 6. The method of any one of claims 1-5,wherein the patient has Group 2 pulmonary hypertension as recognized bythe World Health Organization (WHO).
 7. The method of any one of claims1-6, wherein the patient has pulmonary hypertension due to heart failurewith preserved left ventricular ejection fraction (LVEF).
 8. The methodof any one of claims 1-6, wherein the patient has pulmonary hypertensiondue to heart failure with reduced left ventricular ejection fraction(LVEF).
 9. The method of any one of claims 1-6, wherein the patient hasvalvular heart disease.
 10. The method of any one of claims 1-6, whereinthe patient has congenital/acquired cardiovascular conditions leading topost-capillary PH.
 11. The method of any one of claims 1-5, wherein thepatient has Group 5 pulmonary hypertension as recognized by the WHO. 12.The method of any one of claims 1-5 and 11, wherein the patient haspulmonary hypertension with unclear and/or multifactorial mechanisms.13. The method of claim 9, wherein the valvular heart disease is aorticregurgitation.
 14. The method of claim 9, wherein the valvular heartdisease is aortic stenosis.
 15. The method of claim 9, wherein thevalvular heart disease is mitral valve regurgitation.
 16. The method ofclaim 9, wherein the valvular heart disease is mitral valve stenosis.17. The method of any one of claims 1-17, wherein the patient has a meanpulmonary arterial pressure (mPAP) selected from the group consistingof: a. an mPAP of at least 20 mmHg; b. an mPAP of at least 25 mmHg; c.an mPAP of at least 30 mmHg; d. an mPAP of at least 35 mmHg; e. an mPAPof at least 40 mmHg; f. an mPAP of at least 45 mmHg; and g. an mPAP ofat least 50 mmHg.
 18. The method of any one of claims 1-17, wherein themethod reduces mPAP in the patient.
 19. The method of any one of claims1-18, wherein the method reduces the mPAP in the patient by at least 10%(e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%).
 20. Themethod of any one of claims 1-18, wherein the method reduces the mPAP byat least 3 mmHg (e.g., at least 3, 5, 7, 10, 12, 15, 20, or 25 mm Hg) inthe patient.
 21. The method of any one of claims 1-20, wherein thepatient has a pulmonary arterial wedge pressure (PAWP) of greater than15 mmHg.
 22. The method of claim 21, wherein the method decreases thePAWP in the patient.
 23. The method of claim 22, wherein the methodreduces the PAWP in the patient by at least 10% (e.g., 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, or at least 50%).
 24. The method of any one ofclaims 1-20, wherein the patient has a left ventricular end diastolicpressure (LVEDP) of greater than 15 mmHg.
 25. The method of claim 24,wherein the method decreases the LVEDP in the patient.
 26. The method ofclaim 24, wherein the method reduces the LVEDP in the patient by atleast 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least50%).
 27. The method of any one of claims 1-4 and 6-26, wherein thepatient has a diastolic pressure gradient (DPG) of less than 7 mmHg. 28.The method of any one of claims 1-3 and 5-26, wherein the patient has aDPG of at least 7 mmHg.
 29. The method of claim 28, wherein the methoddecreases the DPG in the patient.
 30. The method of claim 29, whereinthe method reduces the DPG in the patient by at least 10% (e.g., 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%).
 31. The method ofany one of claims 1-4 and 6-30, wherein the patient has a transpulmonarypressure gradient (TPG) of less than or equal to 12 mm Hg.
 32. Themethod of any one of claims 1-3 and 5-30, wherein the patient has a TPGof greater than 12 mm Hg.
 33. The method of claim 32, wherein the methoddecreases the TPG in the patient.
 34. The method of claim 33, whereinthe method reduces the TPG in the patient by at least 10% (e.g., 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%).
 35. The method ofany one of claims 1-3 and 5-34, wherein the patient has a pulmonaryvascular resistance (PVR) greater than or equal to 3 Wood Units.
 36. Themethod of claim 35, wherein the method decreases the PVR in the patient.37. The method of claim 36, wherein the method reduces the PVR in thepatient by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,or at least 50%).
 38. The method of any one of claims 1-4 and 6-37,wherein the method prevents the progression of IpcPH to CpcPH.
 39. Themethod of any one of claims 1-4 and 6-37, wherein the method reduces thedevelopment of a pre-capillary component of PH.
 40. The method of anyone of claims 1-6 and 8-39, wherein the patient has preserved leftventricular ejection fraction.
 41. The method of claim 40, wherein thepreserved left ventricular ejection fraction is greater than 45%. 42.The method of claim 41, wherein the preserved left ventricular fractionis measured using echocardiography.
 43. The method of any one of claims1-42, wherein the patient has diastolic dysfunction of the leftventricle.
 44. The method of any one of claims 1-42, wherein the patienthas systolic dysfunction of the left ventricle.
 45. The method of anyone of claims 1-44, wherein the method decreases right ventricularhypertrophy in the patient.
 46. The method of claim 45, wherein themethod decreases right ventricular hypertrophy in the patient by atleast 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least50%).
 47. The method of any one of claims 1-46, wherein the methoddecreases left ventricular hypertrophy in the patient.
 48. The method ofclaim 47, wherein the method decreases left ventricular hypertrophy inthe patient by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, or at least 50%).
 49. The method of any one of claims 1-48, whereinthe method decreases smooth muscle hypertrophy in the patient.
 50. Themethod of claim 49, wherein the method decreases smooth musclehypertrophy in the patient by at least 10% (e.g., 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, or at least 50%).
 51. The method of any one ofclaims 1-50, wherein the method decreases pulmonary arteriolemuscularity in the patient.
 52. The method of claim 51, wherein themethod decreases pulmonary arteriole muscularity in the patient by atleast 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least50%).
 53. The method of any one of claims 1-52, wherein the patient hasa right ventricular systolic pressure (RVSP) of greater than 35 mmHg.54. The method of claim 53, wherein the method decreases the RVSP in thepatient.
 55. The method of claim 54, wherein the method reduces the RVSPin the patient by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, or at least 50%).
 56. The method of any one of claims 1-55, whereinthe patient has left ventricular fibrosis.
 57. The method of claim 56,wherein the method decreases the left ventricular fibrosis in thepatient.
 58. The method of claim 57, wherein the method reduces the leftventricular fibrosis in the patient by at least 10% (e.g., 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, or at least 50%).
 59. The method of anyone of claims 1-58, wherein the patient has right ventricular fibrosis.60. The method of claim 59, wherein the method decreases the rightventricular fibrosis in the patient.
 61. The method of claim 60, whereinthe method reduces the right ventricular fibrosis in the patient by atleast 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least50%).
 62. The method of any one of claims 1-61, wherein the patient haspulmonary fibrosis.
 63. The method of claim 62, wherein the methoddecreases the pulmonary fibrosis in the patient.
 64. The method of claim63, wherein the method reduces the pulmonary fibrosis in the patient byat least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least50%).
 65. The method of any one of claims 1-64, wherein the patient hasa comorbidity selected from the group consisting of systemichypertension, diabetes mellitus, obesity, coronary artery disease (CAD),heart failure, and anemia.
 66. The method of any one of claims 1-65,comprising further administering to the patient an additional activeagent and/or supportive therapy.
 67. The method of claim 66, wherein theadditional active agent and/or supportive therapy is selected from thegroup consisting of: beta-blockers, angiotensin-converting enzymeinhibitors (ACE inhibitors), angiotensin receptor blockers (ARBs),neprilysin inhibitors, angiotensin receptor-neprilysin inhibitors(ARNI), mineralocorticoid receptor antagonists (MRA),hyperpolarization-activated cyclic nucleotide-gated (HCN) channelblockers, diuretic agents, lipid-lowering medications, endothelinblockers, PDE5 inhibitors, prostacyclins, cardiac resynchronizationtherapy, valve replacement, valve repair, implantablecardioverter-defibrillator (ICD), or a left ventricular assist device(LVAD).
 68. The method of claim 66, wherein the additional active agentand/or supportive therapy is selected from the group consisting of:prostacyclin and derivatives thereof (e.g., epoprostenol, treprostinil,and iloprost); prostacyclin receptor agonists (e.g., selexipag);endothelin receptor antagonists (e.g., thelin, ambrisentan, macitentan,and bosentan); calcium channel blockers (e.g., amlodipine, diltiazem,and nifedipine); anticoagulants (e.g., warfarin); diuretics; oxygentherapy; atrial septostomy; pulmonary thromboendarterectomy;phosphodiesterase type 5 inhibitors (e.g., sildenafil and tadalafil);activators of soluble guanylate cyclase (e.g., cinaciguat andriociguat); ASK-1 inhibitors (e.g., CIIA; SCH79797; GS-4997;MSC2032964A; 3H-naphtho[1,2,3-de]quiniline-2,7-diones, NQDI-1;2-thioxo-thiazolidines,5-bromo-3-(4-oxo-2-thioxo-thiazolidine-5-ylidene)-1,3-dihydro-indol-2-one);NF-κB antagonists (e.g., dh404, CDDO-epoxide; 2,2-difluoropropionamide;C28 imidazole (CDDO-Im); 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid(CDDO); 3-Acetyloleanolic Acid; 3-Triflouroacetyloleanolic Acid;28-Methyl-3-acetyloleanane; 28-Methyl-3-trifluoroacetyloleanane;28-Methyloxyoleanolic Acid; SZC014; SCZ015; SZC017; PEGylatedderivatives of oleanolic acid; 3-O-(beta-D-glucopyranosyl) oleanolicacid; 3-O-[beta-D-glucopyranosyl-(1→3)-beta-D-glucopyranosyl] oleanolicacid; 3-O-[beta-D-glucopyranosyl-(1→2)-beta-D-glucopyranosyl] oleanolicacid; 3-O-[beta-D-glucopyranosyl-(1→3)-beta-D-glucopyranosyl] oleanolicacid 28-O-beta-D-glucopyranosyl ester;3-O-[beta-D-glucopyranosyl-(1→2)-beta-D-glucopyranosyl] oleanolic acid28-O-beta-D-glucopyranosyl ester;3-O-[a-L-rhamnopyranosyl-(1→3)-beta-D-glucuronopyranosyl] oleanolicacid; 3-O-[alpha-L-rhamnopyranosyl-(1→3)-beta-D-glucuronopyranosyl]oleanolic acid 28-O-beta-D-glucopyranosyl ester;28-O-β-D-glucopyranosyl-oleanolic acid; 3-O-β-D-glucopyranosyl(1→3)-β-D-glucopyranosiduronic acid (CS1); oleanolic acid3-O-β-D-glucopyranosyl (1→3)-β-D-glucopyranosiduronic acid (CS2); methyl3,11-dioxoolean-12-en-28-olate (DIOXOL); ZCVI₄-2; Benzyl3-dehydr-oxy-1,2,5-oxadiazolo[3′,4′:2,3]oleanolate); eplerenone,spironolactone, ivabradine, implantable cardioverter-defibrillator(ICD), a left ventricular assist device (LVAD), or lung and/or hearttransplantation.
 69. The method of any one of claims 1-68, wherein thepatient has elevated brain natriuretic peptide (BNP) levels as comparedto a healthy patient.
 70. The method of claim 69, wherein the patienthas a BNP level of at least 100 pg/mL (e.g., 100, 150, 200, 300, 400,500, 1000, 3000, 5000, 10,000, 15,000, or 20,000 pg/mL).
 71. The methodof claim 69 or claim 70, wherein the method decreases BNP levels in thepatient by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, or at least 80%).
 72. The method of anyone of claims 1-70, wherein the method decreases BNP levels to normallevels (i.e., <100 pg/ml).
 73. The method of any one of claims 1-72,wherein the method decreases NT-proBNP levels in the patient.
 74. Themethod of any one of claims 1-73, wherein the method decreases NT-proBNPlevels in the patient by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%).
 75. Themethod of any one of claims 1-74, wherein the method decreases NT-proBNPlevels in the patient by at least 30%.
 76. The method of any one ofclaims 1-75, wherein the method decreases NT-proBNP levels to normallevels.
 77. The method of claim 76, wherein the normal level ofNT-proBNP is <100 pg/ml.
 78. The method of any one of claims 1-77,wherein the method increases exercise capacity of the patient.
 79. Themethod of any one of claims 1-78, wherein the patient has a 6-minutewalk distance from 150 to 400 meters.
 80. The method of any one ofclaims 1-78, wherein the patient has a 6-minute walk distance from 150to 550 meters.
 81. The method of any one of claims 1-80, wherein themethod increases the patient's 6-minute walk distance.
 82. The method ofany one of claims 1-81, wherein the method increases the patient's6-minute walk distance by at least 10 meters (e.g., at least 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, or more than400 meters).
 83. The method of any one of claims 1-82, wherein themethod reduces the patient's Borg dyspnea index (BDI).
 84. The method ofany one of claims 1-83, wherein the method reduces the patient's BDI byat least 0.5 index points (e.g., at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 index points). 85.The method of any one of claims 1-84, wherein the patient has decreasedrenal function.
 86. The method of any one of claims 1-85, wherein themethod further improves renal function.
 87. The method of any one ofclaims 1-86, wherein the patient has Functional Class II or Class IIIpulmonary hypertension in accordance with the World HealthOrganization's functional classification system for pulmonaryhypertension.
 88. The method of any one of claims 1-86, wherein thepatient has Functional Class I, Class II, Class III, or Class IVpulmonary hypertension as recognized by the World Health Organization.89. The method of any one of claims 1-88, wherein the method prevents ordelays pulmonary hypertension Functional Class progression (e.g.,prevents or delays progression from Functional Class I to Class II,Class II to Class III, or Class III to Class IV pulmonary hypertensionas recognized by the World Health Organization).
 90. The method of anyone of claims 1-88, wherein the method promotes or increases pulmonaryhypertension Functional Class regression (e.g., promotes or increasesregression from Class IV to Class III, Class III to Class II, or ClassII to Class I pulmonary hypertension as recognized by the World HealthOrganization).
 91. The method of any one of claims 1-82, wherein thepatient has Functional Class II or Class III pulmonary hypertension inaccordance with the New York Heart Association's functionalclassification system for pulmonary hypertension.
 92. The method of anyone of claims 1-82, wherein the patient has Functional Class I, ClassII, Class III, or Class IV pulmonary hypertension as recognized by theNew York Heart Association.
 93. The method of any one of claims 1-82,91, and 92, wherein the method prevents or delays pulmonary hypertensionFunctional Class progression (e.g., prevents or delays progression fromFunctional Class I to Class II, Class II to Class III, or Class III toClass IV pulmonary hypertension as recognized by the New York HeartAssociation).
 94. The method of any one of claims 1-82, 91, and 92,wherein the method promotes or increases pulmonary hypertensionFunctional Class regression (e.g., promotes or increases regression fromClass IV to Class III, Class III to Class II, or Class II to Class Ipulmonary hypertension as recognized by the New York Heart Association).95. The method of any one of claims 1-94, wherein the method delaysclinical worsening of PcPH.
 96. The method of claim 95, wherein themethod delays clinical worsening of PcPH in accordance with the WorldHealth Organization's functional classification system for pulmonaryhypertension.
 97. The method of claim 95, wherein the method delaysclinical worsening of PcPH in accordance with the New York HeartAssociation's functional classification system for pulmonaryhypertension.
 98. The method of any one of claims 1-97, wherein themethod reduces the risk of hospitalization for one or more complicationsassociated with PcPH.
 99. The method of any one of claims 1-98, whereinthe patient has a hemoglobin level from >8 and <15 g/dl.
 100. The methodof any one of claims 1-99, wherein the patient has been treated with oneor more vasodilators.
 101. The method of any one of claims 1-100,wherein the patient has been treated with one or more agents selectedfrom the group consisting of: phosphodiesterase type 5 inhibitors,soluble guanylate cyclase stimulators, prostacyclin receptor agonist,and endothelin receptor antagonists.
 102. The method of claim 101,wherein the one or more agents is selected from the group consisting of:bosentan, sildenafil, beraprost, macitentan, selexipag, epoprostenol,treprostinil, iloprost, ambrisentan, and tadalafil.
 103. The method ofany one of claims 1-102, wherein the method further comprisesadministration of one or more vasodilators.
 104. The method of any oneof claims 1-103, wherein the method further comprises administration ofone or more agents selected from the group consisting of:phosphodiesterase type 5 inhibitors, soluble guanylate cyclasestimulators, prostacyclin receptor agonist, and endothelin receptorantagonists.
 105. The method of claim 104, wherein the one or moreagents is selected from the group consisting of: bosentan, sildenafil,beraprost, macitentan, selexipag, epoprostenol, treprostinil, iloprost,ambrisentan, and tadalafil.
 106. The method of any one of claims 1-105,wherein the ActRII polypeptide comprises an amino acid sequence that isat least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of aminoacids corresponding to residues 30-110 of SEQ ID NO:
 1. 107. The methodof any one of claims 1-105, wherein the ActRII polypeptide comprises anamino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto the amino acid sequence SEQ ID NO:
 2. 108. The method of any one ofclaims 1-105, wherein the ActRII polypeptide comprises an amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO:
 3. 109. The method of any one ofclaims 106-108, wherein the ActRII polypeptide is a fusion proteinfurther comprising an Fc domain of an immunoglobulin.
 110. The method ofclaim 109, wherein the Fc domain of the immunoglobulin is an Fc domainof an IgG1 immunoglobulin.
 111. The method of claim 109 or 110, whereinthe Fc fusion protein further comprises a linker domain positionedbetween the ActRII polypeptide domain and the Fc domain of theimmunoglobulin.
 112. The method of claim 111, wherein the linker domainis selected from the group consisting of: TGGG (SEQ ID NO: 20), TGGGG(SEQ ID NO: 18), SGGGG (SEQ ID NO: 22), GGGGS (SEQ ID NO: 22), GGG (SEQID NO: 16), GGGG (SEQ ID NO: 17), and SGGG (SEQ ID NO: 21).
 113. Themethod of any one of claims 1-112, wherein the ActRII polypeptidecomprises an amino acid sequence that is at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to the amino acid sequence of SEQ ID NO:
 23. 114. Themethod of any one of claims 1-105, wherein the polypeptide comprises anamino acid sequence that is at least 90% identical to an amino acidsequence corresponding to residues 30-110 of SEQ ID NO: 1, wherein thepolypeptide binds to activing and/or GDF11.
 115. The method of any oneof claims 1-105, wherein the polypeptide comprises an amino acidsequence that is at least 90% identical to an amino acid sequencecorresponding to residues 21-135 of SEQ ID NO: 1, wherein thepolypeptide binds to activing and/or GDF11.
 116. The method of any oneof claims 1-115, wherein the polypeptide is lyophilized.
 117. The methodof any one of claims 1-116, wherein the polypeptide is soluble.
 118. Themethod of any one of claims 1-117, wherein the polypeptide isadministered using subcutaneous injection.
 119. The method of any one ofclaims 1-118, wherein the polypeptide is administered every 4 weeks.120. The method of any one of claims 1-119, wherein the polypeptide ispart of a homodimer protein complex.
 121. The method of any one ofclaims 1-120, wherein the polypeptide is glycosylated.
 122. The methodof any one of claims 1-121, wherein the polypeptide has a glycosylationpattern obtainable by expression in a Chinese hamster ovary cell. 123.The method of any one of claims 1-122, wherein the ActRII polypeptidebinds to one or more ligands selected from the group consisting of:activin A, activin B, and GDF11.
 124. The method of claim 123, whereinthe ActRII polypeptide further binds to one or more ligands selectedfrom the group consisting of: BMP10, GDF8, and BMP6.
 125. A kitcomprising a lyophilized polypeptide and an injection device, whereinthe polypeptide is an ActRII polypeptide comprising an amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to anamino acid sequence that begins at any one of amino acids 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 of SEQ ID NO: 1 and ends at any one ofamino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135of SEQ ID NO:
 1. 126. The kit of claim 125, wherein the polypeptide is apolypeptide comprising an amino acid sequence that is at least 90%identical to an amino acid sequence corresponding to residues 30-110 ofSEQ ID NO:
 1. 127. The kit of claim 125 or 126, wherein the polypeptideis a polypeptide comprising an amino acid sequence that is at least 95%identical to the amino acid sequence corresponding to residues 30-110 ofSEQ ID NO:
 1. 128. The kit of any one of claims 125-127, wherein thepolypeptide is a polypeptide comprising an amino acid sequence that isat least 99% identical to the amino acid sequence corresponding toresidues 30-110 of SEQ ID NO:
 1. 129. The kit of any one of claims125-128, wherein the polypeptide is a polypeptide comprising the aminoacid sequence corresponding to residues 30-110 of SEQ ID NO:
 1. 130. Thekit of any one of claims 125-129, wherein the polypeptide is apolypeptide consisting of the amino acid sequence corresponding toresidues 30-110 of SEQ ID NO:
 1. 131. The kit of claim 125, wherein thepolypeptide is a polypeptide comprising an amino acid sequence that isat least 90% identical to the amino acid sequence corresponding toresidues 21-135 of SEQ ID NO:
 1. 132. The kit of claim 125 or claim 131,wherein the polypeptide is a polypeptide comprising an amino acidsequence that is at least 95% identical to the amino acid sequencecorresponding to residues 21-135 of SEQ ID NO:
 1. 133. The kit of anyone of claims 125, 131, or 132, wherein the polypeptide is a polypeptidecomprising an amino acid sequence that is at least 99% identical to theamino acid sequence corresponding to residues 21-135 of SEQ ID NO: 1.134. The kit of any one of claim 125 or 131-133, wherein the polypeptideis a polypeptide comprising the amino acid sequence corresponding toresidues 21-135 of SEQ ID NO:
 1. 135. The kit of any one of claim 125 or131-134, wherein the polypeptide is a polypeptide consisting of theamino acid sequence corresponding to residues 21-135 of SEQ ID NO: 1.136. The kit of claim 125, wherein the polypeptide is a polypeptidecomprising an amino acid sequence that is at least 90% identical to theamino acid sequence of SEQ ID NO:
 2. 137. The kit of claim 125 or claim136, wherein the polypeptide is a polypeptide comprising an amino acidsequence that is at least 95% identical to the amino acid sequence ofSEQ ID NO:
 2. 138. The kit of any one of claims 125, 136, or 137,wherein the polypeptide is a polypeptide comprising an amino acidsequence that is at least 99% identical to the amino acid sequence ofSEQ ID NO:
 2. 139. The kit of any one of claim 125 or 136-138, whereinthe polypeptide is a polypeptide comprising the amino acid sequence ofSEQ ID NO:
 2. 140. The kit of any one of claim 125 or 136-139, whereinthe polypeptide is a polypeptide consisting of the amino acid sequenceof SEQ ID NO:
 2. 141. The kit of claim 125, wherein the polypeptide is apolypeptide comprising an amino acid sequence that is at least 90%identical to the amino acid sequence of SEQ ID NO:
 3. 142. The kit ofclaim 125 or claim 141, wherein the polypeptide is a polypeptidecomprising an amino acid sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO:
 3. 143. The kit of any one of claims125, 141, or 142, wherein the polypeptide is a polypeptide comprising anamino acid sequence that is at least 99% identical to the amino acidsequence of SEQ ID NO:
 3. 144. The kit of any one of claim 125 or141-143, wherein the polypeptide is a polypeptide comprising the aminoacid sequence of SEQ ID NO:
 3. 145. The kit of any one of claim 125 or141-144, wherein the polypeptide is a polypeptide consisting of theamino acid sequence of SEQ ID NO:
 3. 146. The kit of any one of claims125-145, wherein the polypeptide is a fusion protein further comprisingan Fc domain of an immunoglobulin.
 147. The kit of claim 146, whereinthe Fc domain of the immunoglobulin is an Fc domain of an IgG1immunoglobulin.
 148. The kit of claim 146 or claim 147, wherein thefusion protein further comprises a linker domain positioned between thepolypeptide domain and the Fc domain of the immunoglobulin.
 149. The kitof claim 148, wherein the linker domain is selected from the groupconsisting of: TGGG (SEQ ID NO: 20), TGGGG (SEQ ID NO: 18), SGGGG (SEQID NO: 19), GGGGS (SEQ ID NO: 22), GGG (SEQ ID NO: 16), GGGG (SEQ ID NO:17), and SGGG (SEQ ID NO: 21).
 150. The kit of claim 148 or claim 149,wherein the linker domain comprises TGGG (SEQ ID NO: 20).
 151. The kitof any one of claims 125-150, wherein the ActRII polypeptide comprisesan amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the amino acid sequence of SEQ ID NO:
 23. 152. The kit ofany one of claims 125-151, wherein the ActRII polypeptide comprises theamino acid sequence of SEQ ID NO:
 23. 153. The kit of any one of claims125-152, wherein the ActRII polypeptide consists of the amino acidsequence of SEQ ID NO:
 23. 154. The kit of any one of claims 125-153,wherein the polypeptide is part of a homodimer protein complex.
 155. Thekit of any one of claims 125-154, wherein the polypeptide isglycosylated.
 156. The kit of any one of claims 125-155, wherein thepolypeptide binds to one or more ligands selected from the groupconsisting of: activin A, activin B, and GDF11.
 157. The kit of claim156, wherein the polypeptide further binds to one or more ligandsselected from the group consisting of: BMP10, GDF8, and BMP6.
 158. Thekit of any one of claims 125-155, wherein the polypeptide binds toactivin and/or GDF11.
 159. The kit of any one of claims 125-158, whereinthe kit comprises one or more vials containing the lyophilizedpolypeptide.
 160. The kit of any one of claims 125-159, wherein theinjection device comprises a pre-filled syringe.
 161. The kit of any oneof claims 125-159, wherein the injection device comprises a pumpapparatus.
 162. The kit of claim 161, wherein the pump apparatuscomprises an electromechanical pumping assembly.
 163. The kit of claim161, wherein the pump apparatus is a wearable pump apparatus.
 164. Thekit of claim 162, wherein the pre-filled syringe comprises areconstitution solution.
 165. The kit of claim 162, wherein thereconstitution solution comprises a pharmaceutically acceptable carrierand/or excipient.
 166. The kit of claim 165, wherein thepharmaceutically acceptable carrier is selected from saline solution,purified water, or sterile water for injection.
 167. The kit of claim165, wherein the pharmaceutically acceptable excipient is selected froma buffering agent [e.g., citric acid (monohydrate) and/or trisodiumcitrate (dehydrate)], a surfactant (e.g., polysorbate 80), a stabilizer(e.g., sucrose), and a lyoprotectant (e.g., sucrose).
 168. The kit ofany one of claims 125-167, wherein the injection device comprises a vialadapter.
 169. The kit of claim 168, wherein the vial adapter is capableof attaching to a vial.
 170. The kit of claim 168 or claim 169, whereinthe vial adapter is capable of attaching to a pre-filled syringe. 171.The kit of claim 170, wherein the pre-filled syringe and the vial areattached to opposite ends of the vial adapter.
 172. The kit of claim171, wherein the reconstitution solution is transferred from thepre-filled syringe to the vial.
 173. The kit of claim 125-172, whereinthe lyophilized polypeptide is reconstituted into a sterile injectablesolution.
 174. The kit of claim 125-172, wherein the lyophilizedpolypeptide is reconstituted into a sterile injectable solution prior touse.
 175. The kit claim of 173 or claim 174, wherein the sterileinjectable solution is sterile water for injection.
 176. The kit of anyone of claims 173-175, wherein the sterile injectable solution isadministered parenterally.
 177. The kit of claim 176, wherein theinjection device is used to administer the sterile injectable solutionparenterally.
 178. The kit of any one of claims 173-177, wherein thesterile injectable solution is administered via subcutaneous injection.179. The kit of any one of claims 173-177, wherein the sterileinjectable solution is administered via intradermal injection.
 180. Thekit of any one of claims 173-177, wherein the sterile injectablesolution is administered via intramuscular injection.
 181. The kit ofany one of claims 173-177, wherein the sterile injectable solution isadministered via intravenous injection.
 182. The kit of any one ofclaims 173-177, wherein the sterile injectable solution isself-administered.
 183. The kit of any one of claims 125-182, whereinthe sterile injectable solution comprises a therapeutically effectivedose.
 184. The kit of claim 183, wherein the therapeutically effectivedose comprises a weight based dose.
 185. The kit of any one of claims125-184, wherein the lyophilized polypeptide is administered every 4weeks.
 186. The kit of any one of claims 125-185, wherein the kit isused to treat post-capillary pulmonary hypertension (PcPH).
 187. The kitof claim 186, wherein the PcPH is isolated post-capillary pulmonaryhypertension (IpcPH).
 188. The kit of claim 186, wherein the PcPH iscombined post- and pre-capillary PH (CpcPH).
 189. The kit of any one ofclaims 186-188, wherein the patient has Group 2 pulmonary hypertensionas recognized by the WHO.
 190. The kit of any one of claims 186-189,wherein the patient has pulmonary hypertension due to heart failure withpreserved left ventricular ejection fraction (LVEF).
 191. The kit of anyone of claims 186-189, wherein the patient has pulmonary hypertensiondue to heart failure with reduced left ventricular ejection fraction(LVEF).
 192. The kit of any one of claims 186-189, wherein the patienthas valvular heart disease.
 193. The kit of any one of claims 186-189,wherein the patient has congenital/acquired cardiovascular conditionsleading to post-capillary PH.
 194. The kit of any one of claims 186-188,wherein the patient has Group 5 pulmonary hypertension as recognized bythe WHO.
 195. The kit of any one of claims 186-188, wherein the patienthas pulmonary hypertension with unclear and/or multifactorialmechanisms.
 196. The kit of any one of claims 1-195, wherein the shelflife of the lyophilized polypeptide is at least 1, 1.5, 2, 2.5, or 3years.
 197. The kit of any one of claims 1-195, wherein the lyophilizedpolypeptide is reconstituted.
 198. The kit of claim 197, wherein thereconstituted polypeptide has a shelf life of at least 2 hrs, 3 hrs, or4 hrs.